ES2963759T3 - 5'-nucleotidase modulators, ecto and their use - Google Patents

Abstract

Disclosed herein are compounds that modulate the conversion of AMP to adenosine by 5'-nucleotidase, ecto, and compositions containing the compounds and methods for synthesizing the compounds. Also provided is the use of said compounds and compositions for the treatment and/or prevention of a wide range of diseases, disorders and conditions, including cancer and immune-related disorders, which are mediated by 5'-nucleotidase, ecto. (Automatic translation with Google Translate, without legal value)

Description

description

5'-nucleotidase modulators, ecto and their use

field

[0001] Provided herein, for example, are compounds and compositions for the Inhibition of Adenosine by 5'-nucleotide, ecto, also known as CD73, and pharmaceutical compositions comprising the same. Also provided herein, for example, are methods of treating or preventing a disease, disorder or condition, or a symptom thereof, mediated by the inhibition of adenosine by 5'-nucleotidase, ecto. Any reference in the description to treatment methods refers to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method of treating the human (or animal) body by therapy (or for diagnosis).

background of the invention

[0002] Purinergic signaling, a type of extracellular signaling mediated by purine nucleotides and nucleosides such as ATP and adenosine, involves the activation of purinergic receptors in the cell and/or in nearby cells, resulting in the regulation of cellular functions. Most cells have the capacity to release nucleotides, which generally occurs through regulated exocytosis (see Praetorius, H.<a>.; Leipziger, J. (1 March 2010) Ann Rev Physiology 72(1): 377-393). The released nucleotides can then be hydrolyzed extracellularly by a series of cell membrane-bound enzymes called ectonucleotidases.

[0003] Ectonucleotides catalyze the conversion of ATP to adenosine, an endogenous modulator that influences multiple systems, including the immune system, cardiovascular system, central nervous system, and respiratory system. Adenosine also promotes fibrosis in various tissues. In the first stage of adenosine production, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also known as CD39 (Cluster of Differentiation 39), hydrolyzes ATP to ADP, and then ADP to AMP. In the next step, AMP is converted to adenosine by 5'-nucleotidase, ecto (NT5E or 5NT), also known as CD73 (Cluster of Differentiation 73).

[0004] The enzymatic activities of CD39 and CD73 play strategic roles in calibrating the duration, magnitude and chemical nature of purinergic signals sent to various cells (e.g., immune cells). Alteration of these enzymatic activities can change the course or dictate the outcome of various pathophysiological events, such as cancer, autoimmune diseases, infections, atherosclerosis, and ischemia-reperfusion injury, suggesting that these ectoenzymes represent new therapeutic targets. to treat various disorders.

[0005] Inhibition of CD73 with monoclonal antibodies, RNA<s>I or small molecules delays tumor growth and metastasis (Stagg, J. (2010) PNAS U.S.A. 107:1547-52). For example, treatment with anti-CD73 antibodies was shown to inhibit the growth and metastasis of breast tumors in animal models (Stagg, J. (26 Jan 2010) PNAS U.S.A, 107(4): 1547-52). Additionally, the use of antibodies that specifically bind to CD73 has been evaluated for the treatment of bleeding disorders (eg, hemophilia) (USP 9,090,697). Recently, several efforts have been made to develop therapeutically useful small molecular inhibitors of CD73. For example, Bhattarai et al. ((2015) J Med Chem 58:6248-63) have studied derivatives and analogues of a,p-methylene-ADP (<a>O<p>CP), one of the most metabolically stable, potent and selective CD73 inhibitors that are known, and purine CD73 derivatives have been reported in the patent literature (WO 2015/164573). However, the development of small molecules has been hampered due to, for example, less than ideal metabolic stability.

[0006] Bhattarai et al (J. Med. Chem. 2015, 58, 15, 6248-6263) discloses ecto-5'-Nucleotidase (eN, CD73) inhibitors and their potential use as anticancer therapeutics. The eN inhibitor a,p-methylene-ADP (AOPCP, adenosine-5'-(9-[(phosphonomethyl)phosphonic acid]) was used as the backbone, and derivatives modified at various positions were prepared.

[0007] Furtmann et al (BioorgMed Chem. 2013 No<v>1;21(21): 6616-22) discloses a series of ecto-5'-nucleotidase (e5NT, CD73) inhibitors based on calculations of model-based coupling.

[0008] In view of the role played by CD73 in cancer, as well as in a diverse range of other diseases, disorders and conditions, and the current lack of CD73 inhibitors available to clinicians, new CD73 inhibitors, and compositions, are needed. and methods associated with them.

brief summary of the invention

[0009] The present invention relates to compounds that modulate the conversion of AMP to adenosine by 5'-nucleotidase, ecto (NT5E or 5NT; also known as CD73), and compositions (for example, pharmaceutical compositions) comprising the compounds . Said compounds, including methods for their synthesis, and compositions are described in detail below.

[0010] Consequently, in a first aspect, the present invention relates to compounds having the formula:

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein,

each R<1> is independently selected from the group consisting of hydrogen, optionally substituted Ci -C6 alkyl, optionally substituted aryl and -C(R<2>R<2>)-0-C (0)-0R<3>, or two R<1>groups are optionally combined to form a 5- to 7-membered ring;

each R<2>is independently selected from the group consisting of H and optionally substituted C1-C6 alkyl;

each R<3>is independently selected from the group consisting of H, CrCa alkyl and optionally substituted aryl;

R<5>is selected from the group consisting of H and optionally substituted C1-C6 alkyl;

X is selected from the group consisting of O, CH2 and S;

A is selected from the group formed by:

each of which is optionally substituted by between 1 and 5 R6 substituents, and wherein the subscript n is an integer between 0 and 3;

Z is selected from the group consisting of O, CH2, CHR6 and NR6;

each R6 is independently selected from the group consisting of OH, H, CH3, CN, F, optionally substituted C1-C6 alkyl and 0C(0)-C1-C6 alkyl; and optionally two R6 groups at adjacent ring vertices are joined to form a 5- to 6-membered ring having at least one heteroatom as a ring vertex; and

Het is selected from the group consisting of:

Ra is selected from the group consisting of H, NH2, NHR<7>, NHC(0)R<7>, NR<7>R<7>, R<7>, OH, SR<7>and OR<7> ; Rb is selected from the group consisting of H, halogen, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, and OR<7>; Rc is selected from the group consisting of H, halogen, haloalkyl, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, OR<7>, SR<7>, SO2R7, - X <1> -NH2, -X1_NHR <7>, -X <1> -NR7R <7>, -X <1> -OH, -X <1> -OR <7>, -X <1> -SR <7>and -X<1>-S0<2>R<7>; Re is selected from the group consisting of H, halogen and optionally substituted C<i>-C<6>alkyl;

each X<1>is C1-C4 alkylene; and

each R<7>is independently selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C2-C10 alkenyl, optionally substituted C2-C10 alkyne, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted cycloheteroalkyl 4-7 membered optionally substituted, cycloheteroalkylCi -C4alkyl optionally substituted 4-7 membered optionally substituted aryl, arylCi -C4alkyl optionally substituted, arylC2-C4alkenyl optionally substituted, arylC2-C4alkynyl optionally substituted, heteroaryl optionally substituted, heteroarylCi -C4alkyl optionally substituted , optionally substituted heteroarylCi-C4alkenyl, optionally substituted heteroarylC2-C4alkynyl and, optionally, two R<7> groups attached to a nitrogen atom join to form a 4- to 7-membered heterocyclic ring, optionally fused to an aryl ring.

[0011]In a second aspect, the present invention relates to a pharmaceutical composition comprising a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable excipient.

[0012]In a third aspect, the present invention relates to a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in a method of treating a disease, disorder or condition, mediated by less in part by CD73; optionally, wherein said compound is administered in an amount effective to reverse or stop the progression of D73-mediated immunosuppression.

[0013]In a fourth aspect, the present invention relates to a combination comprising a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one additional therapeutic agent; optionally, wherein the at least one additional therapeutic agent is:

(i) a chemotherapeutic agent, an immunomodulatory and/or anti-inflammatory agent, an antihypercholesterolemia agent or an anti-infective agent; either

(ii) an immune checkpoint inhibitor.

[0014]In a fifth aspect, the present invention relates to a kit comprising a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one additional therapeutic agent; optionally in which

(i) the at least one additional therapeutic agent is a chemotherapeutic agent, an immunomodulatory and/or anti-inflammatory agent, an antihypercholesterolemia agent or an anti-infective agent; either

(ii) the at least one additional therapeutic agent is an immune checkpoint inhibitor.

[0015]In a sixth aspect, the present invention relates to a compound of the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in a method of treating cancer in a subject, said method comprising administering to said subject an effective amount of the compound and an immune checkpoint inhibitor; optionally:

(i) wherein said administration is prior to, simultaneous with, or subsequent to radiotherapy; I

(ii) wherein said compound and said immune checkpoint inhibitor are administered in combination; either

(iii) wherein said compound and said immune checkpoint inhibitor are administered sequentially; either

(I<v>) wherein said compound is administered after said immune checkpoint inhibitor; either

(v) wherein said compound is administered before said immune checkpoint inhibitor.

[0016]The present invention also relates to the use of such compounds and compositions for the treatment and/or prevention of a diverse range of diseases, disorders and conditions mediated, in whole or in part, by CD73. CD73 inhibitors have been linked to the treatment of various disorders, including cancer, fibrosis, neurological and neurodegenerative disorders (e.g., depression and Parkinson's disease), ischemic brain and heart diseases, disorders related to the immune system and disorders with an inflammatory component. [See, for example, Sorrentino et al (2013) Oncolmmunol, 2:e22448, doi: 10.4161/onc¡.22448; and Regateiro et al. (2012) Clin. Exp. Immunol, 171:1-7]. In particular embodiments, the compounds described herein act to inhibit the immunosuppressive activity and/or the anti-inflammatory activity of CD73, and are useful as therapeutic or prophylactic therapy when such inhibition is desired. Unless otherwise indicated, when the uses of the compounds of the present invention are described herein, it will be understood that said compounds may be in the form of a composition (for example, a pharmaceutical composition).

[0017]As used herein, the terms "CD73 inhibitor", "CD73 blocker", "adenosine 5'-nucleotidase inhibitor, ecto", "NT5E inhibitor", "inhibitor of 5NT" and all other related terms accepted in the art refer to a compound capable of modulating, directly or indirectly, the CD73 receptor in an in vitro assay, an in<v>¡<v>o model, and/or others means indicative of therapeutic efficacy. The<s>terms also refer to compounds that show at least some therapeutic benefit in a human subject.

[0018]Although the compounds of the present invention are believed to effect their activity by inhibiting CD73, a precise understanding of the underlying mechanism of action of the compounds is not required to practice the invention. For example, Io<s>compounds may also effect their activity, at least in part, through modulation (e.g., inhibition) of other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling system consists of transporters, enzymes and receptors responsible for the synthesis, release, action and extracellular inactivation of (mainly) ATP and its extracellular breakdown product adenosine (Sperlagh, B. et al. (Dec 2012) Neuropsychopharmacologia Hungarica 14 (4):231-38). Since inhibition of CD73 causes a decrease in adenosine, Io<s>CD73 inhibitors can be used for the treatment of diseases or disorders mediated by adenosine and its<s>actions on Io<s>adenosine receptors, including A1 ,A2a, A2b and A3. [See Yegutkin, GG (May 2008) Biochimica Biophysica Acta 1783(5):673-94].

[0019]For purposes of the present disclosure, the purinergic signaling process can be described as comprising the following components. The purinergic receptors (P1, P2X and P2Y), a first component, are membrane receptors that mediate various physiological functions (for example, relaxation of intestinal smooth muscle) in response to the release of ATP or adenosine; In general, all cells have the capacity to release nucleotides into the extracellular medium, frequently through regulated exocytosis. Nucleoside transporters (NTs), a second component, are membrane transport proteins that transport nucleoside substrates (e.g., adenosine) across cell membranes; The extracellular concentration of adenosine can be regulated by Ios NT, possibly in the form of a feedback loop that connects receptor signaling with transporter function. As described previously, ectonucleotidases (CD73 and CD39) hydrolyze Io<s>nucleotides released into the extracellular medium and constitute another component. Another component of the purinergic signaling process comprises pannexins; in particular, the pannexin-1 (PANX1) channel is an integral component of the purinergic P2X/P2Y signaling pathway and the key contributor to the pathophysiological release of At P.

[0020]In some embodiments, the present invention contemplates compounds for use in the treatment or prevention of cancer in a subject (e.g., a human) comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor. described in this document. The present invention includes methods of treating or preventing a cancer in a subject by administering to the subject a CD73 inhibitor in an amount effective to reverse or stop the progression of CD73-mediated immunosuppression. In some embodiments, CD73-mediated immunosuppression is mediated by an antigen-presenting cell (APC).

[0021]Examples of cancers that can be treated with the compounds and compositions described here include, among other cancers of the prostate, colorectal, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testicles, head, neck, skin (including melanoma and basal cell carcinoma), mesothelial lining white blood cells (including lymphoma and leukemia) esophagus, breast, muscle, connective tissue, lung (including small cell lung carcinoma and non-small cell carcinoma), adrenal gland, thyroid, kidney or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basal cell carcinoma and testicular seminoma. In some embodiments of the present invention, the cancer is melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumor, lymphoma, sarcoma, ovarian cancer, or Kaposi's sarcoma. Candidate cancers to be treated with the compounds of the present invention are described below.

[0022]The present invention contemplates compounds for use in the treatment of a subject receiving a bone marrow transplant or a peripheral blood stem cell transplant by administering a therapeutically effective amount of a CD73 inhibitor sufficient to increase the delayed hypersensitivity reaction to tumor antigen, delay the time to post-transplant malignancy relapse, increase post-transplant relapse-free survival time and/or increase long-term post-transplant survival.

[0023]In certain embodiments, the present invention contemplates compounds for use in the treatment or prevention of an infectious disorder (e.g., a viral infection) in a subject (e.g., a human) comprising administering to the subject a therapeutically administered amount effective of at least one CD73 inhibitor (e.g., a novel inhibitor of the present invention). In some embodiments, the infectious disorder is a viral infection (e.g., a chronic viral infection), a bacterial infection, a fungal infection, or a parasitic infection. In certain embodiments, the viral infection is human immunodeficiency virus or cytomegalovirus.

[0024]In other embodiments, the present invention contemplates compounds for use in the treatment and/or prevention of diseases, disorders and conditions related to immunity; diseases that have an inflammatory component; as well as disorders associated with the above; with at least one CD73 inhibitor of the present invention. Examples of diseases, disorders and conditions related to the immune system are described below.

[0025]Other diseases, disorders and conditions that can be treated or prevented, in whole or in part, by modulating CD73 activity are candidate indications for the CD73 inhibitor compounds of the present invention.

[0026]The present invention further contemplates the use of the CD73 inhibitors described herein in combination with one or more additional agents. One or more additional agents may have some CD73 modulatory activity and/or may function through distinct mechanisms of action. In some embodiments, such agents comprise radiation (e.g., localized radiation therapy or total body radiation therapy) and/or other treatment modalities of a non-pharmacological nature. When a combination therapy is used, the CD73 inhibitor(s) and the additional agent(s) may be presented as a single composition or multiple compositions, and the treatment modalities may be administered concurrently, sequentially, or by some other regimen. By way of example, the present invention contemplates a treatment regimen in which a radiation phase is followed by a chemotherapeutic phase. Combination therapy may have an additive or synergistic effect. Other advantages of combination therapy are described below.

[0027]In some embodiments, the present invention further comprises the use of the CD73 inhibitors described herein in combination with bone marrow transplantation, peripheral blood stem cell transplantation, other types of transplant therapy.

[0028]In particular embodiments, the present invention contemplates the use of the inhibitors of CD73 function described herein in combination with immune checkpoint inhibitors. Blockade of immune checkpoints, which results in amplification of antigen-specific T cell responses, has proven to be a promising approach in human cancer therapeutics. Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various tumor cell types, that are candidates for blockade include PD1 (programmed cell death protein 1); PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic T lymphocyte-associated antigen 4); TIM3 (T cell membrane protein 3); LAG3 (lymphocyte activation gene 3); A2aR (adenosine A2a receptor A2a); and death inhibitory receptors. Immune checkpoint inhibitors and combination therapy with them are discussed in detail in this document.

[0029]In other embodiments, the present invention provides compounds for use in the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one chemotherapeutic agent, such agents include , but are not limited to alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulfonate esters such as busulfan; nucleoside analogues (e.g., gemcitabine); nitroso ureas such as carmustine, lomustine and streptozocin; topoisomerase 1 inhibitors (e.g. irinotecan); platinum complexes such as cisplatin and carboplatin; bioreducing alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand breaking agents (eg, bleomycin); topoisomerase II inhibitors (for example, amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide and teniposide); DNA minor groove binding agents (eg, plicamycin); antimetabolites (for example, folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacytidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine , fludarabine, pentostatin; asparaginase and ribonucleotide reductase inhibitors such as hydroxyurea); tubulin-interacting agents (eg, vincristine, estramustine, vinblastine, docetaxol, epothilone derivatives and paclitaxel); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbesterol; chlortrianisene; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone ); adrenal corticosteroids (for example, prednisone, dexamethasone, methylprednisolone, and prednisolone); leutinizing hormone-releasing agents or gonadotropin-releasing hormone antagonists (for example, leuprolide acetate and goserelin acetate); and antihormonal antigens (for example, tamoxifen, antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide). The present invention also contemplates the use of CD73 inhibitors in combination with other agents known in the art (for example, arsenic trioxide) and other chemotherapeutic agents that may be developed in the future.

[0030]In some embodiments of compounds for use in the treatment of cancer, administration of a therapeutically effective amount of a CD73 inhibitor in combination with at least one chemotherapeutic agent results in a cancer survival rate greater than cancer survival rate observed by administering either agent alone. In other embodiments of compounds for use in the treatment of cancer, administration of a therapeutically effective amount of a CD73 inhibitor in combination with at least one chemotherapeutic agent results in a reduction in tumor size or a slowing of tumor growth greater than the reduction in tumor size or tumor growth observed by administration of either agent alone.

[0031] In other embodiments, the present invention contemplates compounds for use in the treatment or prevention of cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one transduction inhibitor signals (ITS). In a particular embodiment, the at least one STI is selected from the group consisting of bcr/abl kinase inhibitors, epidermal growth factor (EGF) receptor inhibitors, her-2/neu receptor inhibitors, and farnesyl inhibitors. transferase (FTI). Other candidate CTI agents are described herein.

[0032] The present invention also contemplates compounds for inaugurating the rejection of tumor cells in a subject comprising the administration of a CD73 inhibitor together with at least one chemotherapeutic agent and/or radiotherapy, wherein the resulting rejection of tumor cells is greater than that obtained by administering the CD73 inhibitor, the chemotherapeutic agent or radiotherapy alone.

[0033] In other embodiments, the present invention provides compounds for use in the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one immunomodulator other than a CD73 inhibitor. CD73.

[0034] The present invention contemplates embodiments comprising compounds for use in the treatment or prevention of an infectious disorder (e.g., a viral infection) in a subject (e.g., a human) comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and a therapeutically effective amount of an anti-infective agent or agents, such as one or more antimicrobial agents.

[0035] In other embodiments, treatment of an infectious disorder is effected by coadministration of a vaccine in combination with administration of a therapeutically effective amount of a CD73 inhibitor of the present invention. In some embodiments, the vaccine is an antiviral vaccine, including, for example, an HIV vaccine. In other embodiments, the vaccine is effective against tuberculosis or malaria. In other embodiments, the vaccine is a tumor vaccine (e.g., an effective vaccine against melanoma); The tumor vaccine may comprise genetically modified tumor cells or a genetically modified cell line, including genetically modified tumor cells or a genetically modified cell line that has been transfected to express granulocyte-macrophage stimulating factor (GM-C SF). In certain embodiments, the vaccine includes one or more immunogenic peptides and/or dendritic cells.

[0036] In certain embodiments of compounds for use in treating an infection by administering a CD73 inhibitor and at least one additional therapeutic agent, a symptom of infection observed after administering both the CD73 inhibitor and the therapeutic agent Additional improvement over the same infection symptom observed after administering either alone. In some embodiments, the observed symptom of infection may be reduced viral load, increased CD4+ T cell count, decreased opportunistic infections, increased survival time, eradication of chronic infection, or a combination. thereof.

brief description of the drawings

[0037] Figure 1 shows a simplified representation of extracellular purinergic signaling.

detailed description

[0038] Before the present invention is described in more detail, it should be understood that the invention is not limited to the particular embodiments set forth herein, and it should also be understood that the terminology used herein is for the purpose only. to describe particular embodiments, and is not intended to be limiting.

[0039] When a range of values is provided, it is understood that each intermediate value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other value declared or intermediate in that declared range, is included within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the invention, subject to any limits specifically excluded in the indicated range. Where the indicated range includes one or both limits, ranges excluding one or both of the included limits are also included in the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains.

[0040] It should be noted that, as used herein and in the accompanying claims, the singular forms "a", and "the" include a plural reference unless the context clearly indicates otherwise. It should also be noted that claims can be drafted in a way that excludes any optional elements. As such, this statement is intended to serve as a prior basis for the use of exclusive terminology such as "solely", "only" and the like in connection with the statement of the elements of the claim, or the use of a "negative" limitation.

[0041]The publications discussed herein are provided solely for disclosure prior to the date of submission of this application. Additionally, the release dates provided may be different from the actual release dates, which may require independent confirmation.

General

[0042]The number of subjects diagnosed with cancer and the number of deaths attributable to cancer continue to increase. Traditional treatment approaches, including chemotherapy and radiation therapy, are often difficult for the patient to tolerate and lose effectiveness as cancers (e.g., tumors) evolve to evade such treatments. Recent experimental evidence indicates that CD73 inhibitors may represent an important new therapeutic modality for the treatment of cancer (e.g., breast).

[0043]Promising data also support the role of inhibitors of CD73 function in inhibiting the anti-inflammatory activity of CD73 and/or the immunosuppressive activity of CD73, and therefore CD73 inhibitors may be useful to treat, e.g. immunosuppressive diseases (for example, HIV and AIDS). CD73 inhibition may also be an important treatment strategy for patients with neurological or neuropsychiatric diseases or disorders such as depression.

[0044]The present invention relates, among other things, to small molecule compounds with D73 inhibitory activity, as well as compositions thereof, and to compounds and compositions for use in the treatment and prevention of diseases , disorders and conditions described herein.

Definitions

[0045]Unless otherwise indicated, the following terms have the meanings indicated below. Other terms are defined elsewhere in the specification.

[0046]The term "alkyl", by itself or as part of another substituent, means, unless otherwise indicated, a straight-chain or branched hydrocarbon radical, having the designated number of carbon atoms. C-i-8 means one to eight carbons). Some examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

[0047]The term "cycloalkyl" refers to hydrocarbon rings that have the indicated number of ring atoms (e.g.,<c>3-6 cycloalkyl) and that are fully saturated or that have no more than one double bond between the vertices of the ring. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.

[0048]The term "cycloheteroalkyl" refers to a cycloalkyl ring having the indicated number of vertices (or members) of the ring and having one to five heteroatoms selected from N, O and S, substituting one to five of the carbon vertices, and wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. The cycloheteroalkyl may be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of cycloheteroalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide , piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine and the like. A cycloheteroalkyl group can be attached to the rest of the molecule through a ring carbon or a heteroatom. When "optionally substituted" is used to describe any of the terms "cycloheteroalkyl" or "cycloheteroalkyl-alkyl", it refers to those groups in which the cycloheteroalkyl or alkyl moiety is optionally substituted as in the following definitions referring to the moiety I rent. For example, an optionally substituted cycloheteroalkyl-alkyl group may be optionally substituted on either or both of the cycloheteroalkyl and alkyl moieties, as in the definitions of alkyl substituents below.

[0049]As used herein, a wavy line, "'JVW", that crosses a single, double or triple bond in any chemical structure represented herein, represents the point of attachment of the single, double bond or triple to the rest of the molecule. Additionally, a bond extending to the center of a ring (e.g., a phenyl ring) indicates binding to any of the ring's available vertices. One skilled in the art will understand that multiple substituents shown attached to a ring will occupy ring vertices that provide stable and otherwise sterically compatible compounds. In the case of a divalent component, a representation is understood to include either orientation (direct or reverse). For example, the group "-C(0)NH-" is understood to include a bond in any orientation: -C(0)NH- or -NHC(O)--, and similarly, "-O-CH2CH2 -" is understood to include both -O-CH2CH2- and -CH2CH2-O-.

[0050]The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an amylno group or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl moieties can be the same or different and can also combine to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as dialkylamino or -NRaRb is understood to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

[0051] The terms "arylalkyl" and "heteroarylalkyl" are used in their conventional sense, and refer to those groups in which an aryl group or a heteroaryl group is attached to the rest of the molecule via an alkylene linker -<c>4. An exemplary embodiment of "arylalkyl" is phenylmethyl (or benzyl). Likewise, an exemplary embodiment of "heteroarylalkyl" is, for example, 3-pyridylpropyl. When "optionally substituted" is used to describe either of the terms "arylalkyl" or "heteroarylalkyl", it refers to those groups in which the aryl or heteroaryl moiety is optionally substituted as defined below, and the alkyl moiety is optionally substituted. as defined below.

[0052] The terms "halo" or "halogen", by themselves or as part of another substituent, mean, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom. Additionally, terms such as "haloalkyl" include monohaloalkyl and polyhaloalkyl. For example, the term "CW haloalkyl" includes trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl and the like.

[0053] The term "aryl" means, unless otherwise indicated, a polyunsaturated hydrocarbon group, typically aromatic, which may be a single ring or multiple rings (up to three rings) fused or covalently linked. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl.

[0054] The term "heteroaryl" refers to aryl groups (or rings) containing one to five heteroatoms selected from N, O and S, in which the nitrogen and sulfur atoms are optionally oxidized, and the atom or atoms nitrogen are optionally quaternized. A heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, inyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl , pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, benzofuranoyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and similar. The substituents on a heteroaryl ring may be selected from the group of acceptable substituents described below.

[0055] The above terms (e.g., "alkyl", "aryl", and "heteroaryl"), in some embodiments, will be optionally substituted. The substituents selected for each type of radical are indicated below.

[0056] Optional substituents for alkyl radicals (including those groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) may be a variety of groups selected from: halogen, -OR', -NR'R", - SR', -SiR'R"R'", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR "C(0)R', -NR'-C(0)NR"Rm, -NR"C(0 )2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(0)R', -S(0 )2R', -S(0 )2NR'R", -NR'S(0 )2R", -CN and -NO2 in a number between zero and (2 m' 1), m' being the total number of carbon atoms of said radical. R', R" and Rm each independently refer to hydrogen, unsubstituted C1-8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C1-8 alkyl, C1.8 alkoxy or C1 thioalkoxy groups. 8, or unsubstituted C1-4alkyl groups. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0057] Likewise, the optional substituents for the aryl and heteroaryl groups are varied and are generally selected from: -halogen, -OR', -0C(0)R', -NR'R", -SR', - R', -CN, -NO2, - CO2R', -CONR'R", -C(0)R', -0C(0)NR'R", -NR"C(0)R', -NR" C(0)<2>R', -NR'-C(0)NR"Rm, -NH-C(NH<2>)=NH, -NR'C(NH<2>)=NH, -NH -C(NH<2>)=NR', -S(0)R', - S(0)<2>R', -S(0 )<2>NR'R", -NR'S(0 )< 2>R", -N3, perfluoro(c1-c4)alkoxy and perfluoro(C1-C4)alkyl, in a number between zero and the total number of open valencies in the aromatic ring system; and where R', R" and R'" are independently selected from hydrogen, C3-6 alkyl, CI-s haloalkyl, C2-8 cycloalkyl, C2-8 alkenyl and C2-8 alkynyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of 1-4 carbon atoms.

[0058] Do<s>of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted by a substituent of the formula -T-C(O)-(CH2)<q>-U-, where T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted by a substituent of the formula -A-(CH2)<r>-B-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(0 )<2> -, -S(0 )<2>NR'- or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted with a substituent of the formula -(CH2)s-X-(CH2)t-, where s and t are independent integers from 0 to 3, and -O-, -NR'-, -S-, -S(O)-, -S(0 )<2>-, or -S(0 )<2>NR'-. The substituent R' in -NR'- and -S(0 )<2>NR' is selected from hydrogen or unsubstituted C1-6 alkyl.

[0059] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (S).

[0060] The term "pharmaceutically acceptable salts" includes salts of active compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired base, either pure or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, natural amines and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired acid, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydrodiodic, or phosphoric and the like, as well as salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic and similar acids. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galactunoric acids and the like (see, for example, Berge, S.M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted to basic or acidic addition salts.

[0061] Neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the original compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0062] Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with an enzyme. or suitable chemical reagent.

[0063] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0064] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are intended to be encompassed within the scope of the present invention. When a stereochemical representation is shown, it refers to the compound in which one of the isomers is present and substantially free of the other isomer. Substantially free of another isomer indicates at least an 80/20 ratio of the two isomers, more preferably 90/10, or 95/5 or more. In some embodiments, one of the isomers will be present in an amount of at least 99%.

[0065] The compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more of the atoms that constitute said compounds. The unnatural proportions of an isotope can be defined from the amount found in nature to an amount consisting of 100% of the atom in question. For example, compounds may incorporate radioactive isotopes, such as tritium (3H), iodine-125 (125l), or carbon-14 (14C), or non-radioactive isotopes, such as deuterium (2H) or carbon-13 (13C). Such isotopic variations may provide additional utilities to those described elsewhere in this application. For example, isotopic variants of the compounds of the invention may find additional utility, including but not limited to, as diagnostic and/or imaging reagents, or as cytotoxic/radiotoxic therapeutic agents. Furthermore, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics that may contribute to improved safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

[0066] The<s>terms "patient" or "subject" are used interchangeably to refer to a human being or a non-human animal (e.g., a mammal).

[0067]The terms "administration", "administer" and the like, when applied, for example, to a subject, cell, tissue, organ or biological fluid, refer to the contact of, for example, a CD73 inhibitor a composition pharmaceutical comprising it or a diagnostic agent with the subject, cell, tissue, organ or biological fluid. In the context of a cell, administration includes contact (e.g., in vitro or ex vivo) of a reagent with the cell, as well as contact of a reagent with a fluid, when the fluid is in contact with the cell. cell.

[0068]The terms "treat'1, "treat", treatment" and the like refer to a course of action (such as the administration of a CD73 inhibitor or a pharmaceutical composition comprising it) initiated after the has diagnosed, observed or similar a disease, disorder or condition, or a symptom thereof, in order to eliminate, reduce, suppress, mitigate or improve, temporarily or permanently, at least one of the underlying causes of a disease, disorder or condition affecting a subject, or at least one of the symptoms associated with a disease, disorder or condition affecting a subject. Therefore, treatment includes inhibition (e.g., stopping the development or further development of the disease, disorder or condition or the clinical symptoms associated with them) of an active disease.

[0069]The term "treatment need," as used herein, refers to the judgment made by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is based on a number of factors that fall within the scope of the doctor or caregiver.

[0070]The terms "prevent", "avoid", "prevention" and the like refer to a course of action (such as the administration of a CD73 inhibitor or a pharmaceutical composition comprising it) initiated in a manner (e.g. ., before the appearance of a disease, disorder, condition or symptom thereof) in order to prevent, suppress, inhibit or reduce, temporarily or permanently, the risk of a subject of developing a disease, disorder, condition or similar (determined, for example, by the absence of clinical symptoms) or delaying their onset, generally in the context of a subject predisposed to suffering from a particular disease, disorder or condition. In certain cases, the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting the progression thereof to a harmful or otherwise unwanted state.

[0071]The term "in need of prevention," as used herein, refers to a judgment made by a physician<u>other caregiver that a subject requires or will benefit from preventive care. This judgment is based on a number of factors that fall within the purview of the doctor or caregiver.

[0072]The phrase "therapeutically effective amount" refers to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition and whether in a single dose or as part of a series of doses, in a quantity capable of having any detectable positive effect on any symptom, aspect or characteristic of a disease, disorder or condition when administered to the subject. The therapeutically effective amount may be determined by measuring the relevant physiological effects, and may be adjusted in relation to the dosage regimen and diagnostic analysis of the subject's condition, and the like. By way of example, measurement of the serum level of a CD73 inhibitor (or, for example, a metabolite thereof) at a given time after administration may be indicative of whether a therapeutically effective amount has been used.

[0073]The phrase "in an amount sufficient to effect a change" means that there is a detectable difference between a level of an indicator measured before (e.g., a reference level) and after the administration of a particular therapy. Indicators include any objective (eg, serum concentration) or subjective (eg, the subject's sense of well-being) parameters.

[0074]The term "small molecules" refers to chemical compounds that have a molecular weight of less than about 10kDa, less than about 2kDa, or less than about IkDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules containing a radioactive atom, and synthetic molecules. Therapeutically, a small molecule may be more permeable to cells, less susceptible to degradation, and less likely to provoke an immune response than large molecules.

[0075]The terms "inhibitors" and "antagonists", or "activators" and "agonists" refer to inhibitory or activating molecules, respectively, for example, for the activation of, for example, a ligand, receptor, cofactor, gene , cell, tissue<u>organ. Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or downregulate, for example, a gene, protein, ligand, receptor, or cell. Activators are molecules that enhance, activate, facilitate, enhance activation, sensitize, or upregulate, for example, a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks or inactivates a constitutive activity. An "agonist" is a molecule that interacts with a target to cause or promote increased activation of the target. An "antagonist" is a molecule that opposes the action(s) of an agonist. An antagonist prevents, reduces, inhibits or neutralizes the activity of an agonist, and an antagonist may also prevent, inhibit or reduce the constitutive activity of a target, for example, a target receptor, even when there is no identified agonist.

[0076]The terms "modulate", "modulation" and the like refer to the ability of a molecule (e.g., an activator or an inhibitor) to increase or decrease the function or activity of CD73, either directly or indirectly. . A modulator may act alone or may use a cofactor, for example, a protein, metal ion, or small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules. There are numerous small molecule compound libraries on the market (e.g., combinatorial libraries) that can serve as a starting point for identifying a modulator. The skilled artisan is capable of developing one or more assays (e.g., biochemical or cellular assays) in which said compound libraries can be screened in order to identify one or more compounds having the desired properties; Subsequently, the expert medicinal chemist is able to optimize said one or more compounds, for example, by synthesizing and evaluating analogues and derivatives thereof. Synthetic and/or molecular modeling studies can also be used in the identification of an Activator.

[0077]The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or a receptor; to the catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation or maturation; to antigenic activity; to the modulation of the activities of other molecules; and the like. The term "proliferative activity" encompasses an activity that promotes, is necessary for, or is specifically associated with, for example, normal cell division, as well as cancer, tumors, dysplasia, cellular transformation, metastasis and angiogenesis.

[0078]As used herein, "comparable", "comparable activity", "comparable activity to", "comparable effect", "comparable effect to" and the like are relative terms that may be considered quantitatively and/or qualitatively. . The meaning of terms often depends on the context in which they are used. As an example, two agents that both activate a receptor may be considered to have a comparable effect from a qualitative perspective, but the two agents may be considered to lack a comparable effect from a quantitative perspective if one agent is only able to achieve 20 % of the activity of the other agent as determined in a state-of-the-art assay (e.g., dose-response assay) or in a state-of-the-art animal model. When comparing one result with another (e.g., a result with a reference norm), "comparable" often (but not always) means that a result deviates from a reference norm by less than 35%, by less of 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3 %, less than 2% or less than 1%. In certain embodiments, a result is comparable to a reference standard if it deviates less than 15%, less than 10%, or less than 5% from the reference standard. By way of example, but not limitation, activity or effect may refer to efficacy, stability, solubility or immunogenicity.

[0079]"Substantially pure" indicates that a component represents more than 50% of the total content of the composition, and typically more than 60% of the total content. More typically, "substantially pure" refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the component of interest will represent more than 90% or more than 95% of the total content of the composition.

[0080]The term "response", for example, of a cell, tissue, organ or organism, encompasses a change in biochemical or physiological behavior, for example, concentration, density, adhesion or migration within a biological compartment, rate of gene expression or differentiation state, where the change is correlated with activation, stimulation or treatment, or with internal mechanisms such as genetic programming. In certain contexts, the terms "activation", "stimulation" and the like refer to cellular activation regulated by internal mechanisms, as well as external or environmental factors; while the terms "inhibition", "downregulation" and the like refer to opposite effects.

5'-Nucleotidase, ecto and inhibition thereof

[0081]Human CD73 (also called 5'-nucleotidase, ecto; NT5E; or 5NT) is a protein of 574 amino acid residues (accession No. AAH6593). Eukaryotic CD73 functions as a non-covalent homodimer with two structural domains, in which the N- and C-terminal domains are connected by a hinge region that allows the enzyme to undergo large domain movements and switch between open and closed conformations (Knapp , K. et al. (2012) Structure 20:2161-73).

[0082]As used herein, the terms "CD73 inhibitor", "CD73 blocker", "adenosine 5'-nucleotidase inhibitor, ecto", "NT5E inhibitor", "5NT inhibitor" and all other related terms accepted in the art refer to a compound capable of modulating, directly or indirectly, the CD73 receptor in an in vitro assay, an in vivo model, and/or other means indicative of therapeutic efficacy. The<s>terms also refer to compounds that show at least some therapeutic benefit in a human subject. A CD73 inhibitor can be a competitive, non-competitive or irreversible inhibitor of<c>D73. "A competitive CD73 inhibitor" is a compound that reversibly inhibits the activity of the CD73 enzyme at the catalytic site; "a non-competitive CD73 inhibitor" is a compound that reversibly inhibits the activity of the CD73 enzyme at a non-catalytic site; and "an irreversible CD73 inhibitor" is a compound that irreversibly eliminates the activity of the CD73 enzyme by forming a covalent bond (<u>another stable means of inhibiting enzyme function) with the enzyme.

[0083]CD73 inhibitors can modulate purinergic signaling, a type of extracellular signaling mediated by purine nucleotides and nucleosides such as ATP and adenosine. Purinergic signaling involves the activation of purinergic receptors on the cell and/or nearby cells, resulting in the regulation of cellular functions. The enzymatic activity of CD73 plays a strategic role in calibrating the duration, magnitude and chemical nature of purinergic signals sent to various cells (e.g., immune cells). Alteration of these enzymatic activities can change the course or dictate the outcome of various pathophysiological events, such as cancer, autoimmune and inflammatory diseases, infections, atherosclerosis, and ischemiareperfusion injury, suggesting that these ectoenzymes represent new therapeutic targets. to treat various disorders.

[0084] Studies using CD73-overexpressing tissues and using CD73knock-out mice have provided evidence that CD73 inhibitors have potential utility for melanomas, lung cancer, prostate cancer, and breast cancer (see , for example, Sadej R. (2006) Melanoma Res 16:213-22). Since higher levels of CD73 expression are associated with tumor neovascularization, invasiveness, resistance to chemotherapy, and metastasis, CD73 inhibitors can be used to control tumor progression and metastasis. Other potential utilities are discussed in this document.

[0085]As discussed above, although the compounds of the present invention are believed to exert their activity by inhibiting CD73, a precise understanding of the underlying mechanism of action of the compounds is not required to practice the invention. For example, compounds may also exert their activity, at least in part, through modulation (e.g., inhibition) of other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling system consists of transporters, enzymes and receptors responsible for the synthesis, release, action and extracellular inactivation of (mainly) ATP and its extracellular breakdown product adenosine (Sperlagh, B. et al. (Dec 2012) Neuropsychopharmacologia Hungarica 14 (4):231-38). Figure 1 shows a simplified representation of extracellular purinergic signaling (see, for example, North RA (Oct 2002) Physiological Reviews 82(4):1013-67). As indicated there, there are several potential opportunities for modulation of the signaling process. However, as an expert can see, some of these opportunities are easier to take advantage of than others.

Identification of CD73 inhibitors with desirable characteristics

[0086]The present invention is directed, in part, to the identification of CD73 inhibitors with at least one property or characteristic that is of therapeutic relevance. Candidate inhibitors may be identified using, for example, a test or model accepted in the art, examples of which will be apparent to the skilled craftsman. The assay used to determine the CD73 inhibitory activity of the compounds described here is set forth in the Experimental section.

[0087]After identification, candidate inhibitors can be further evaluated by using techniques that provide data relating to the characteristics of the inhibitors (e.g., pharmacokinetic parameters). Comparisons of candidate inhibitors to a reference standard (which may be the “best in class” of current inhibitors) are indicative of the potential viability of such candidates.

[0088]CD73 inhibitors that may serve as target or reference compounds include a,p-methylene-ADP (AOPCP) and its<s>derivatives and analogs described by Bhattarai et al. ((2015) J Med Chem 58:6248-63) and purine CD73 derivatives reported in PCT Publn. 2015/164573. Other reference compounds subsequently identified by the expert may also be used to evaluate the feasibility of candidate CD73 inhibitors.

Compounds of the invention

[0089]Compounds of formula (I) are included herein:

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein,

each R<1> is independently selected from the group consisting of hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R<2>R<2>)-0-C (0)-0R<3>, or two R<1>groups are optionally combined to form a 5- to 7-membered ring;

each R<2>is independently selected from the group consisting of H and optionally substituted C1-C6 alkyl;

each R<3>is independently selected from the group consisting of H, C1-C6 alkyl and optionally substituted aryl;

R5 is selected from the group consisting of H and optionally substituted Ci-C6 alkyl;

X is selected from the group consisting of O, CH2 and S;

A is selected from the group formed by:

each of which is optionally substituted by between 1 and 5 R6 substituents, and wherein the subscript n is an integer between 0 and 3;

Z is selected from the group consisting of CH2, CHR6, NR6, and O;

each R6 is independently selected from the group consisting of H, CH3, OH, CN, F, optionally substituted C1-C6 alkyl and 0 C(0 )-C1-C6 alkyl; and optionally two R6 groups at adjacent ring vertices are joined to form a 5- to 6-membered ring having at least one heteroatom as a ring vertex; and

Het is selected from the group consisting of:

in which the wavy line indicates the point of union with the rest of the compound, and in which:

Ra is selected from the group consisting of H, NH2, NHR<7>, NHC(0)R<7>, NR<7>R<7>, R<7>, OH, SR<7>and OR<7> ; Rb is selected from the group consisting of H, halogen, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, and OR<7>; Rc is selected from the group consisting of H, halogen, haloalkyl, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, OR<7>, SR<7>, SO2R7, - X <1> -NH2, -X1_NHR <7>, -X <1> -NR7R <7>, -X <1> -OH, -X <1> -OR <7>, -X <1> -SR <7>and -X<1>-S0<2>R<7>; Re is selected from the group consisting of H, halogen and optionally substituted C1-C6 alkyl; each X<1>is C1-C4 alkylene; and

each R<7>is independently selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C2-C10 alkenyl, optionally substituted C2-C10 alkyne, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted cycloheteroalkyl 4-7 membered optionally substituted, cycloheteroalkylC1-C4alkyl optionally substituted 4-7 membered alkyl, optionally substituted aryl, arylC1-C4alkyl optionally substituted, arylC2-C4alkenyl optionally substituted, arylC2-C4alkynyl optionally substituted, heteroaryl optionally substituted, heteroarylC1-C4alkyl optionally substituted , optionally substituted heteroarylC1-C4alkenyl, optionally substituted heteroarylC2-C4alkynyl and, optionally, two R<7> groups attached to a nitrogen atom join to form a 4- to 7-membered heterocyclic ring, optionally fused to an aryl ring.

[0090] In the above formula, the term "optionally substituted" is used in relation to alkyl groups, cycloalkyl groups, cycloheteroalkyl groups, aryl groups and heteroaryl groups. Within each of these groups, some selected optional substituents are as follows:

Alkyl groups: halogen, -OR', -NR'R", -SR', -SiR'R"Rm, -0C(0)R', -C(0)R', -CO2R', - CONR'R ", -0C(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"Rm, -NR"C(0 )<2>R', -CN and -NO2. R', R" and Rm each independently refer to hydrogen, unsubstituted C1-4 alkyl, or C1.4 haloalkyl. When R' and R" are bonded to the same nitrogen atom, or when R" and Rm are bonded to the same nitrogen, they can combine with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

Cycloalkyl groups and cycloheteroalkyl groups: The selected substituents noted above for Io<s>"alkyl groups" are also useful with Io<s>ddoalkyl and cycloheteroalkyl groups. Additionally, each of Io<s>ddoalkyl and cycloheteroalkyl groups may be optionally substituted with oxo (=0).

Aryl groups and heteroaryl groups: -halogen, -0R', -0C(0)R', -NR'R", -R', -CN, -NO2, - CO2R', -CONR'R", -C( 0)R', -0C(0)NR'R", -NR"C(0)R', -NR"C(0)2R', -NR'-C(0)NR"Rm, - S( 0)2R', -S(0)2NR'R", -NR'S(0)2R", and perfluoro(Ci-C4)alkyl, where R', R" and Rm are independently selected from hydrogen, C-m alkyl, haloalkyl C1.4 and C3-6 ddoalkyl.

[0091] In a selected group of embodiments, compounds of Formula (I) are provided where A has the formula:

which is optionally replaced with 1 to 5 R6

[0092] In another selected group of embodiments, compounds of Formula (I) are provided wherein A has a formula selected from the group consisting of:

[0093]In some selected embodiments, any of a l to a16 may be combined independently with any of b l to b9, to provide selected embodiments of Formula (I). For example, compounds of Formula (l) are provided having the following combinations of Het-A-: a l/b l; a1/b2; a1/b3; a1/b4; a1/b5; a1/b6; a1/b7; a1/b8; a1/b9 a2/b1 a2/b2 ; a2/b3; a2/b4; a2/b5 a2/b6; a2/b7; a2/b8; a2/b9; a3/b1; a3/b2; a3/b3; a3/b4 a3/b5 a3/b6; a3/b7; a3/b8 a3/b9 a4/b1 ; a4/b2; a4/b3; a4/b4; a4/b5; a4/b6; a4/b7; a4/b8; a4/b9; a5/b1; a5/b2; a5/b3 a5/b4 a5/b5; a5/b6; a5/b7 a5/b8 a5/b9 a6/b1 a6/b2; a6/b3; a6/b4; a6/b5; a6/b6; a6/b7; a6/b8; a6/b9; a7/b1; a7/b2 a7/b3 a7/b4; a7/b5; a7/b6 a7/b7 a7/b8 a7/b9 a8/b1; a8/b2 a8/b3; a8/b4; a8/b5; a8/b6; a8/b7; a8/b8; a8/b9; a9/b1 a9/b2 a9/b3; a9/b4; a9/b5; a9/b6; a9/b7; a9/b8; a9/b9; a10/b1; a10/b3 ; a10/b4; a10/b5; a10/b6; a10/b7; a10/b8; a10/b9 a ll /b l ; a11/b2; a11/b3; a11/b4; a11/b5; a11/b6; a11/b7; a11/b8; a11/b9; a12/b1; a12/b2; a12/b3; a12/b4; a12/b5; a12/b6 a12/b7; a12/b8; a12/b9; a13/b1; a13/b2; a13/b3; a13/b4; a13/b5; a13/b6; a13/b7; a13/b8; a13/b9; a14/b1; a14/b2; a14/b3 a14/b4; a14/b5; a14/b6; a14/b7; a14/b8; a14/b9; a15/b1; a15/b2; a15/b3; a15/b4; a15/b5; a15/b6; a15/b7; a15/b8; a15/b9 a16/b1; a16/b2; a16/b3; a16/b4; a16/b5; a16/b6; a16/b7; a16/b8; or a16/b9.

[0094] Also provided herein, in a group of embodiments, are compounds having the formula:

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein,

each R1 is independently selected from the group consisting of hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R2R2)-0-C(0)-0R3, or two R1 groups are optionally combined to form a ring of 5 to 7 members;

each R2 is independently selected from the group consisting of H and optionally substituted C1-C6 alkyl;

each R3 is independently selected from the group consisting of H, Ci-C6 alkyl and optionally substituted aryl;

R5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl;

X is O;

A is selected from the group formed by:

AND

Het is selected from the group consisting of:

in which the wavy line indicates the point of union with the rest of the compound, and in which:

Ra is selected from the group consisting of H, NH2, NHR<7>, NHC(0)R<7>, NR<7>R<7>, R<7>, OH, SR<7>and OR<7> ; Rb is selected from the group consisting of H, halogen, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, and OR<7>; Rc is selected from the group consisting of H, halogen, haloalkyl, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, OR<7>, SR<7>, SO2R7, - X<1>-NH2, -X<1>-NHR<7>, -X<1>-NR7R<7>, -X<1>-OH, -X<1>-OR<7>, – <1>-SR<7>and -X<1>-S0<2>R<7>; Re is selected from the group consisting of H, halogen and optionally substituted C1-C6 alkyl; each X<1>is C1-C4 alkylene; AND

each R<7>is independently selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C2-C10 alkenyl, optionally substituted C2-C10 alkyne, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted cycloheteroalkyl 4-7 membered optionally substituted, cycloheteroalkylC1-C4alkyl optionally substituted 4-7 membered alkyl, optionally substituted aryl, arylC1-C4alkyl optionally substituted, arylC2-C4alkenyl optionally substituted, arylC2-C4alkynyl optionally substituted, heteroaryl optionally substituted, heteroarylC1-C4alkyl optionally substituted , optionally substituted heteroarylC1-C4alkenyl, optionally substituted heteroarylC2-C4alkynyl and, optionally, two R<7> groups attached to a nitrogen atom join to form a 4- to 7-membered heterocyclic ring, optionally fused to an aryl ring.

[0095] In a selected group of embodiments, the compounds of formula (IVa) are those in which A is

[0096] In another selected group of embodiments, the compounds of formula (IVa) are those in which Het is:

Synthesis Methods

[0097]In general, the compounds provided herein can be prepared by conventional methods as described in the Examples below.

Modifications to Improve Inhibitor Characteristics

[0098]It is often beneficial, and sometimes imperative, to improve one or more physical properties of the treatment modalities disclosed herein and/or the manner in which they are administered. Improvements in physical properties include, for example, methods of increasing water solubility, bioavailability, serum half-life and/or therapeutic half-life; and/or the modulation of biological activity.

Therapeutic and Prophylactic Uses

[0099]The present invention contemplates the use of the CD73 inhibitors described herein in the treatment or prevention of a wide range of diseases, disorders and/or conditions, and/or the symptoms thereof. Although particular uses are described in detail below, it should be understood that the present invention is not so limited. In addition, although general categories of particular diseases, disorders and conditions are set forth below, some of the diseases, disorders and conditions may fall into more than one category, and others may not fall into any of the disclosed categories.

[0100]Oncology-Related Disorders. In accordance with the present invention, a CD73 inhibitor can be used to treat or prevent a proliferative condition or disorder, including a cancer, for example, cancer of the uterus, cervix, breast, prostate, testicles, gastrointestinal tract (e.g. , esophagus, oropharynx, stomach, small or large intestine, colon or rectum), kidney, renal cell, bladder, bone, bone marrow, skin, head or neck, liver, gallbladder, heart, lung, pancreas, salivary gland, gland adrenal, thyroid, brain (e.g., gliomas), lymph nodes, central nervous system (CNS), and peripheral nervous system (PNS), and cancers of the hematopoietic system and immune system (e.g., spleen or thymus). The present invention also provides methods for treating or preventing other cancer-related diseases, disorders or conditions, including, for example, immunogenic tumors, non-immunogenic tumors, latent tumors, virus-induced cancers (e.g., epithelial cell cancers). , endothelial cell cancers, squamous cell carcinomas and papillomaviruses), adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas, teratocarcinomas, chemically induced cancers, metastasis and angiogenesis. The invention contemplates reducing tolerance to a tumor cell or cancer cell antigen, for example, by modulating the activity of a regulatory T cell and/or a CD8+ T cell (see, for example, Ramírez-Montagut, et al. (2003) Oncogene 22:3180-87; and Sawaya, et al. (2003) New Engl. J. Med. 349:1501-09). In certain embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma or leukemia. The use of the terms cancer-related diseases, disorders and conditions generally refers to conditions associated, directly or indirectly, with cancer, and includes, for example, angiogenesis and precancerous conditions such as dysplasia.

[0101]In certain embodiments, a cancer may be metastatic or at risk of becoming metastatic, or may occur in diffuse tissue, including cancers of the blood or bone marrow (e.g., leukemia). In some other embodiments, the compounds of the invention can be used to overcome T cell tolerance.

[0102] In some embodiments, the present invention provides compounds for use in the treatment of a proliferative condition, cancer, tumor or precancerous condition with a CD73 inhibitor and at least one additional therapeutic or diagnostic agent, examples of which are set forth elsewhere in this document.

[0103]Immune-Related Disorders and Disorders with an Inflammatory Component. As used herein, terms such as "immune disease", "immune condition", "immune disorder", "inflammatory disease", "inflammatory condition", "inflammatory disorder" and the like broadly encompass any condition related to the immune system (e.g., an autoimmune disease) or a disorder with an inflammatory component that can be treated with the CD73 inhibitors described herein so that some therapeutic benefit is obtained. These conditions are often inextricably intertwined with other diseases, disorders and conditions. By way of example, an "immune condition" may refer to proliferative conditions, such as cancer, tumors, and angiogenesis; including infections (acute and chronic), tumors and cancers that resist eradication by the immune system.

[0104]The CD73 inhibitors of the present invention can be used to increase or improve an immune response; to improve immunization, including increasing the effectiveness of vaccines; and to increase inflammation. Immunodeficiencies associated with immunodeficiency diseases, immunosuppressive medical treatments, acute and/or chronic infections and aging can be treated with the compounds described here. CD73 inhibitors can also be used to stimulate the immune system of patients suffering from iatrogenically induced immunosuppression, including those who have undergone bone marrow transplants, chemotherapy or radiotherapy.

[0105]In particular embodiments of the present disclosure, CD73 inhibitors are used to increase or enhance an immune response to an antigen by providing adjuvant activity. In a particular embodiment, at least one antigen or vaccine is administered to a subject in combination with at least one CD73 inhibitor of the present invention to prolong an immune response to the antigen or vaccine. Also provided are therapeutic compositions that include at least one antigenic agent or vaccine component, including, but not limited to, viruses, bacteria and fungi, or portions thereof, proteins, peptides, tumor-specific antigens and nucleic acid vaccines, in combination with at least one CD73 inhibitor of the present invention.

[0106] Microbe-Related Disorders, By inhibiting the immunosuppressive and anti-inflammatory activity of CD73, the present invention contemplates the use of the CD73 inhibitors described herein in the treatment and/or prevention of any viral, bacterial, fungal disease, disorder or condition , parasitic or other infectious disease for which treatment with a CD73 inhibitor may be beneficial. Examples of such diseases and disorders include HIV and AIDS, staphylococcal and streptococcal infections (e.g., Staphylococcus aureus and streptococcus sanguinis, respectively), leishmania, toxoplasma, trichomonas, giardia, candida albicans, bacillus anthracis and pseudomonas aeruginosa. The invention can be used to treat sepsis, reduce or inhibit bacterial growth and reduce or inhibit inflammatory cytokines,

[0107]Neurological and CNS-Related Disorders. Inhibition of CD73 may also be an important treatment strategy for patients with neurological, neuropsychiatric, neurodegenerative<u>other diseases, disorders and conditions that have some relationship with the central nervous system, including disorders associated with impaired function cognitive and motor function. Examples include Parkinson's disease, extrapyramidal syndrome (EPS), dystonia, akathisia, tardive dyskinesia, restless legs syndrome (RLS), epilepsy, periodic limb movements during sleep (PLMS). , attention deficit disorders, depression, anxiety, dementia, Alzheimer's disease, multiple sclerosis, cerebral ischemia, hemorrhagic stroke, subarachnoid hemorrhage and traumatic brain injury.

[0108]Other Disorders. Embodiments of the present invention contemplate administering the CD73 inhibitors described herein to a subject for the treatment or prevention of any other disorder that may benefit from at least some level of CD73 inhibition. Such diseases, disorders and conditions include, for example, cardiovascular (e.g., cardiac ischemia), gastrointestinal (e.g., Crohn's disease), metabolic (e.g., diabetes), hepatic (e.g., ., liver fibrosis, NASH and NAFLD), pulmonary (e.g., COPD and asthma), ophthalmologic (e.g., diabetic retinopathy), and renal (e.g., kidney failure).

[0109]In some embodiments, the CD73 inhibitors of the present invention can be used to inhibit statin-induced adenosine production, or reduce or diminish increases in blood glucose caused by a statin in a subject taking a statin (p . e.g., lovastatin and pravastatin).

Pharmaceutical Compositions

[0110]The CD73 inhibitors of the present invention may be in the form of compositions suitable for administration to a subject. Generally, such compositions are "pharmaceutical compositions" comprising a CD73 inhibitor(s) and one or more pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients. In certain embodiments, the CD73 inhibitors are present in a therapeutically acceptable amount. The pharmaceutical compositions can be used in the methods of the present invention; Thus, for example, the pharmaceutical compositions may be administered ex vivo or in vivo to a subject to practice the therapeutic and prophylactic methods and uses described herein.

[0111]The pharmaceutical compositions of the present invention may be formulated to be compatible with the intended method or route of administration; Exemplary routes of administration are set forth herein. Furthermore, the pharmaceutical compositions may be used in combination with other therapeutically active agents or compounds described herein to treat or prevent the diseases, disorders and conditions contemplated by the present invention.

[0112]Pharmaceutical compositions containing the active ingredient (e.g., an inhibitor of CD73 function) may be in a form suitable for oral use, for example, as tablets, capsules, tablets, lozenges, aqueous suspensions. >oily, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microspheres or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and said compositions may contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents and preservative agents with the in order to provide pharmaceutically elegant and palatable preparations. Tablets, capsules and the like contain the active ingredient mixed with pharmaceutically acceptable non-toxic excipients that are suitable for the manufacture of tablets. These excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.

[0113]Tablets, capsules and the like suitable for oral administration may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide sustained action. For example, a retarding material such as glyceryl monostearate or glyceryl distearate may be used. They may also be coated by techniques known in the art to form controlled release osmotic therapeutic tablets. Additional agents include biodegradable or biocompatible particles or a polymeric substance such as polyesters, polyamine acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethyl vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers or ethyl vinyl acetate copolymers in order to control the administration of an administered composition. For example, the oral agent may be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, using hydroxymethylcellulose or gelatin microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloidal drug delivery system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, microbeads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. The<s>methods for preparing the aforementioned formulations will be apparent to those skilled in the art.

[0114]Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules. in which the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin or olive oil.

[0115]The aqueous suspensions contain the active materials in a mixture with excipients suitable for their manufacture. Such excipients may be suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, for example a natural phosphatide (e.g. lecithin), or condensation products of an alkylene oxide with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., for heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or products of condensation of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives.

[0116]Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweeteners, such as those mentioned above, and flavorings can be added to obtain a palatable oral preparation.

[0117]Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0118]The pharmaceutical compositions of the present invention may also be presented in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or mixtures of these. Suitable emulsifying agents may be natural gums, for example, gum acacia or gum tragacanth; natural phosphatides, for example, soy, lecithin and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.

[0119]Pharmaceutical compositions typically comprise a therapeutically effective amount of a CD73 inhibitor contemplated by the present invention and one or more pharmaceutically and physiologically acceptable formulation agents. Pharmaceutically or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl , p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents and/or adjuvants. For example, a suitable carrier may be a physiological saline solution or a citrate-buffered saline solution, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases or mixtures thereof. By way of example, the buffer components may be water-soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid and salts thereof. Acceptable buffering agents include, for example, a Tris buffer, N-(2-hydroxyethN)piperazine-N-(2-ethanesulfonic acid) (HEPE), 2-(N-morpholino)ethanesulfonic acid (MES). ), sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS) and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0120]Once a pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form, in a lyophilized form requiring reconstitution before use, in a liquid form requiring dilution before use, or in another acceptable form. In some embodiments, the pharmaceutical composition is supplied in a single-use container (e.g., a single-use vial, ampoule, syringe, or autoinjector (similar, e.g., to an EpiPen®)), while in other embodiments A multi-use container (for example, a multi-use vial) is supplied.

[0121]Formulations may also include carriers to protect the composition against rapid degradation or clearance from the organism, such as a controlled release formulation, including liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a retarding material such as glyceryl monostearate or glyceryl stearate may be used alone, or in combination with a wax. Any drug delivery apparatus can be used to administer a CD73 inhibitor, including implants (e.g., implantable pumps) and catheter systems, pumps and slow injection devices, all of which are well known to the skilled artisan.

[0122]Depot injections, which are generally administered subcutaneously or intramuscularly, can also be used to release the CD73 inhibitors described herein over a defined period of time. Depot injections are typically solid or oily based and typically comprise at least one of the formulation components discussed here. One skilled in the art is familiar with the possible formulations and uses of depot injections.

[0123]The pharmaceutical compositions can be presented in the form of a sterile injectable aqueous or oily suspension. This suspension can be formulated according to the known art using the suitable dispersing or wetting agents and suspending agents mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic diluent or solvent acceptable to the parents, for example, as a solution in 1,3-butane diol. Acceptable diluents, solvents, and dispersion media that may be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor E™ (BAS, Parsippany, NJ), or phosphate-buffered saline (PBS). , ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Furthermore, sterile fixed oils are conventionally used as a solvent or suspending medium. For this, any tasteless fixed oil can be used, including synthetic mono- or diglycerides. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).

[0124]The present invention contemplates the administration of CD73 inhibitors in the form of suppositories for rectal administration. Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at normal temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

[0125]In some embodiments, the CD73 inhibitors contemplated by the present invention may be presented in the form of any other suitable pharmaceutical composition (e.g., aerosols for nasal or inhalation use) currently known or developed in the future.

Routes of Administration

[0126]The present invention contemplates the administration of CD73 inhibitors, and compositions thereof, in any appropriate manner. Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intrapparenchymal), and intracerebroventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal, and inhalation. Depot injections, which are generally administered subcutaneously or intramuscularly, can also be used to release the CD73 inhibitors described herein over a defined period of time.

[0127]Some particular embodiments of the present invention contemplate oral administration.

Combined Therapy

[0128]The present invention contemplates the use of CD73 inhibitors in combination with one or more active therapeutic agents (e.g., chemotherapeutic agents)<u>other prophylactic or therapeutic modalities (e.g., radiation). In this type of combined therapy, the different active agents usually have different and complementary mechanisms of action. Such combination therapy may be especially advantageous by allowing a reduction in the dose of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents. Furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

[0129]As used herein, "combination" includes therapies that can be administered separately, for example, formulated separately for administration separately (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "coformulation").

[0130]In certain embodiments, CD73 inhibitors are administered or applied sequentially, for example, when one agent is administered before one or more agents. In other embodiments, the CD73 inhibitors are administered simultaneously, for example, when two or more agents are administered at the same time or at approximately the same time; The two or more agents may be present in two or more separate formulations or combined in a single formulation (i.e., a coformulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of the present invention.

[0131]The CD73 inhibitors of the present invention can be used in combination with at least one other (active) agent in any manner appropriate to the circumstances. In one embodiment, treatment with at least one active agent and at least one CD73 inhibitor of the present invention is maintained for a period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with a CD73 inhibitor of the present invention is maintained at a constant dosage regimen. . In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with a CD73 inhibitor of the present invention is reduced (e.g., when the subject is stable). lower dose, less frequent dosing, or shorter treatment regimen). In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable), and treatment with the CD73 inhibitor of the present invention is increased (e.g., dosage). higher, more frequent dosing, or longer treatment regimen). In another embodiment, treatment with at least one active agent is maintained and treatment with the CD73 inhibitor of the present invention is reduced or discontinued (e.g., lower dose, less frequent dosing, or shorter treatment regimen). . In another embodiment, treatment with at least one active agent and treatment with the CD73 inhibitor of the present invention are reduced or discontinued (e.g., lower doses, less frequent dosing, or shorter treatment regimen).

[0132]Oncology-Related Disorders. The present invention provides methods of treating and/or preventing a proliferative condition, cancer, tumor or precancerous disease, disorder or condition with a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0133]In certain embodiments, the present invention provides methods for suppressing tumor growth comprising administering a CD73 inhibitor described herein in combination with a signal transduction inhibitor (STI) to achieve additive suppression or synergistic tumor growth. As used herein, the term "signal transduction inhibitor" refers to an agent that selectively inhibits one or more steps of a signaling pathway. Signal transduction inhibitors (STIs) of the present invention include: (i) bcr/abl kinase inhibitors (e.g., GLEEVEC); (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors and antibodies; (iii) her-2/neu receptor inhibitors (e.g., HERCEPTIN); (<iv>) inhibitors of Akt family kinases or the Akt pathway (eg, rapamycin); (<v>) cell cycle kinase inhibitors (e.g., flavopiridol); and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immunomodulation can also be used in combination with the CD73 inhibitors described herein for the suppression of tumor growth in cancer patients.

[0134]Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelanime; nitrogen mustards such as chiorambucil, clomafazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, fenesterin, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, Caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, Marcelomycin, Mitomycins, Mycophenolic Acid, Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Puromycin, Chelamycin, Rhodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as frolinic acid; aceglatone; aldophosphamide glucoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophylinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizophyran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; methotrexate; vincristine; and carboplatin; ; aminopterin; ibadronate; topoisomerase inhibitors (D<m>FO);

[0135]Chemotherapeutic agents also include antihormonal agents that act to regulate or inhibit hormonal action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazoles inhibitors. aromatase, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the combination therapy comprises the administration of a hormone or related hormonal agent.

[0136]Other treatment modalities that may be used in combination with a CD73 inhibitor include radiation therapy, a monoclonal antibody against a tumor antigen, a complex of a monoclonal antibody and a toxin, a T cell adjuvant, bone marrow or cell transplantation. antigen presenting (e.g. dendritic cell therapy).

[0137]Immune Checkpoint Inhibitors. The present invention contemplates the use of the inhibitors of CD73 function described here in combination with immune checkpoint inhibitors.

[0138]The enormous number of genetic and epigenetic alterations characteristic of all Io<s>cancers provides a diverse set of antigens that the immune system can use to distinguish tumor cells from their normal counterparts. In the case of T cells, the final breadth (e.g., Io<s>levels of cytokine production or proliferation) and quality (e.g., the type of immune response generated, such as the pattern of production of cytokines) of the response, which is initiated by antigen recognition by the T cell receptor (TCR), is regulated by a balance between costimulatory and inhibitory signals (immune checkpoints). Under normal physiological conditions, Io<s>immune checkpoints are crucial for the prevention of autoimmunity (i.e., maintenance of self-tolerance) and also for the protection of Io<s>tissues from damage when the immune system responds. to a pathogenic infection. Tumors can deregulate the expression of immune checkpoint proteins as an important mechanism of immune resistance.

[0139]Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various tumor cell types, that are candidates for blockade include PD1 (programmed cell death protein 1) ; PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic T lymphocyte-associated antigen 4); TIM3 (T cell membrane protein 3); LAG3 (lymphocyte activation gene 3); A2aR (adenosine A2a receptor A2a); and killer cell inhibitory receptors, which can be divided into two classes based on their structural characteristics: i) killer cell immunoglobulin-like receptors (KIR), and ii) C-type lectin receptors ( members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described in the literature, including both receptors (e.g., the 2B4 receptor (also known as CD244)) and ligands (e.g., certain inhibitory ligands of the B7 family such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7<x>and VCTN1)). [See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

[0140] The present invention contemplates the use of Io<s>inhibitors of CD73 function described here in combination with inhibitors of the aforementioned immune checkpoint receptors and ligands, as well as immune checkpoint receptors and ligands even to describe. Some immune checkpoint modulators are currently available, while others are in the late stages of development. As an example, when approved for the treatment of melanoma in 2011, the fully humanized CTLA4 monoclonal antibody ipilimumab (YERVOY; Bristol-Myers Squibb) became the first immune checkpoint inhibitor to receive regulatory approval in the US. In the US, fusion proteins comprising CTLA4 and an antibody (CTLA4-Ig; abatcept (ORENCIA; Bristol-Myers Squibb)) have been used for the treatment of rheumatoid arthritis, and other fusion proteins have been shown to be effective in patients with kidney transplant sensitized to Epstein Barr virus. Antibodies against PD1 are being developed (e.g., nivolumab (Bristol-Myers Squibb) and lambrolizumab (Merck)), and anti-PDLI antibodies are also being evaluated (e.g., MPDL3280A (Roche)). Nivolumab has shown promise in patients with melanoma and lung and kidney cancer.

[0141]The present invention encompasses pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0142]Metabolic and Cardiovascular Diseases. The present invention provides compounds for use in methods of treatment and/or prevention of certain cardiovascular and/or metabolic diseases, disorders and conditions, as well as disorders associated therewith, with a CD73 inhibitor and at least one therapeutic or additional diagnosis.

[0143]Examples of therapeutic agents useful in combination therapy for the treatment of hypercholesterolemia (and atherosclerosis as well) include statins (e.g., CRE<s>To R, LESCOL, LIPITOR, MEVACOR, PRAVACOL, and ZOCOR) , which inhibit the enzymatic synthesis of cholesterol; bile acid resins (e.g., COLESTID, LO-CHOLEST, p ReVALITE, Q<u>ESTRAN, and WELCHOL), which sequester cholesterol and prevent its absorption; ezetimibe (ZETIA), which blocks cholesterol absorption; fibric acid (e.g., TRICOR), which lowers Io<s>triglycerides and may modestly increase HDL; niacin (e.g., NIACOR), which modestly reduces LDL cholesterol and Io<s>triglycerides; and/or a combination of the above (e.g., VYTORIN (ezetimibe with simvastatin). Alternative cholesterol treatments that may be candidates for use in combination with the CD73 inhibitors described herein include various supplements and herbs (e.g., garlic, policosanol, and guggul).

[0144]The present invention encompasses pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0145]Immune-related disorders and disorders with an inflammatory component. The present invention provides methods for treating and/or preventing immune-related diseases, disorders and conditions; and diseases, disorders and conditions that have an inflammatory component; with a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0146]Examples of therapeutic agents useful in combination therapy are specific to the underlying disease, disorder or condition, and are known to the skilled artisan.

[0147]Microbial Diseases. The present invention provides methods for treating and/or preventing viral, bacterial, fungal and parasitic diseases, disorders and conditions, as well as disorders associated therewith, with a CD73 inhibitor and at least one additional therapeutic or diagnostic agent (e.g. e.g., one or more antiviral agents and/or one or more agents not associated with viral therapy).

[0148]Such combination therapy includes antiviral agents targeting various stages of the viral life cycle and having different mechanisms of action, including, but not limited to, the following: viral decapping inhibitors (e.g., amantadine and rimantidine ); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents directed against integrase; agents that block the binding of transcription factors to viral DNA; agents (eg, antisense molecules) that affect translation (eg, fomivirsen); agents that modulate translation/ribozyme function; protease inhibitors; modulators of viral assembly (e.g., rifampicin); antiretrovirals, such as nucleoside reverse transcriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC, d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine); nucleotide analogue reverse transcriptase inhibitors; and agents that prevent the release of viral particles (e.g., zanamivir and oseltamivir). Treatment and/or prevention of certain viral infections (e.g., HIV) often requires a group ("cocktail") of antiviral agents.

[0149]Other antiviral agents contemplated for use in combination with a CD73 inhibitor include, but are not limited to, the following abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, bocepreertevirt, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, http://en.wikipedia.org/wiki/Fusion_inhibitor ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, various interferons ( e.g., peginterferon alfa-2a), lopinavir, loviride, maraviroc, moroxidine, metisazone, nelfinavir, nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine and zalcitabine.

[0150]The present invention contemplates the use of the inhibitors of CD73 function described herein in combination with antiparasitic agents. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamniquin, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox and nitroimidazole. The skilled artisan knows other agents that may be useful in the treatment of parasitic disorders.

[0151]Embodiments of the present invention contemplate the use of the CD73 inhibitors described herein in combination with agents useful in the treatment or prevention of bacterial disorders. Antibacterial agents can be classified in several ways, for example, according to their mechanism of action, their chemical structure or their spectrum of activity. Examples of antibacterial agents are those that act on the bacterial cell wall (e.g., cephalosporins and penicillins) or the cell membrane (e.g., polymyxins), or interfere with essential bacterial enzymes (e.g., sulfonamides). , rifamycins and quinolines). Most antibacterial agents targeting protein synthesis (e.g., tetracyclines and macrolides) are bacteriostatic, while agents such as aminoglycosides are bactericidal. Another way to categorize antibacterial agents is based on their specificity; “Narrow-spectrum” agents target specific types of bacteria (e.g., Gram-positive bacteria such as Streptococcus), while “broad-spectrum” agents have activity against a broader range of bacteria. The skilled artisan knows the types of antibacterial agents that are appropriate for use in specific bacterial infections.

[0152]Embodiments of the present invention contemplate the use of the CD73 inhibitors described herein in combination with agents useful in the treatment or prevention of fungal disorders. Antifungal agents include polyenes (e.g., amphotericin, nystatin, and pimaricin); azoles (e.g., fluconazole, itraconazole, and ketoconazole); allylamines (e.g., naftifine and terbinafine) and morpholines (e.g., amorolfine); and antimetabolites (e.g., 5-fluorocytosine).

[0153]The present invention encompasses pharmaceutically acceptable salts, acids or derivatives of the agents (and members of classes of agents) set forth above.

Dosage

[0154]The CD73 inhibitors of the present invention may be administered to a subject in an amount depending, for example, on the purpose of administration (e.g., the degree of resolution desired); the age, weight, sex and health and physical condition of the subject to whom the formulation is administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof. The dosage regimen may also take into account the existence, nature and extent of any adverse effects associated with the agent or agents being administered. Effective dosage amounts and dosage regimens can be readily determined from, for example, safety and dose escalation trials, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.

[0155]In general, dosing parameters dictate that the dosage amount be less than an amount that could be irreversibly toxic to the subject (the maximum tolerated dose (MTD)) and not less than an amount necessary to produce a measurable effect. in the subject. These quantities are determined, for example, by the pharmacokinetic and pharmacodynamic parameters associated with ADME, taking into account the route of administration and other factors.

[0156]An effective dose (ED) is the dose or amount of an agent that produces a desired therapeutic response or effect in some fraction of the subjects who take it. The "median effective dose" or ED50 of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although the ED50 is commonly used as a reasonable expectation measure of the effect of an agent, it is not necessarily the dose that a clinician would consider appropriate taking into account all relevant factors. Thus, in some situations the effective quantity is greater than the calculated ED50, in other situations the effective quantity is less than the calculated ED50, and in other situations the effective quantity is the same as the calculated ED50.

[0157]Further, an effective dose of the CD73 inhibitors of the present invention may be an amount that, when administered in one or more doses to a subject, produces a desired result relative to a healthy subject. For example, for a subject experiencing a particular disorder, an effective dose may be one that improves a diagnostic parameter, measure, marker and the like of that disorder by at least 5%, at least 10%, at least 20%. %, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90%, where 100% is defined as the diagnostic parameter, measurement, marker and the like exhibited by a normal subject.

[0158]In certain embodiments, the CD73 inhibitors contemplated by the present invention can be administered (e.g., orally) at dosage levels of about 0.01 mg/kg to about 50 mg/kg, or about 1 mg/kg to approximately 25 mg/kg, of the subject's body weight per day, one or more times a day, to obtain the desired therapeutic effect.

[0159]For the administration of an oral agent, the compositions may be provided in the form of tablets, capsules and the like containing 1.0 to 1000 milligrams of the active ingredient, in particular 1.0 milligrams.0, 3.0, 5 ,0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0 , 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 milligrams of the active ingredient.

[0160]In certain embodiments, the dose of the desired CD73 inhibitor is contained in a "unit dosage form." The phrase "unit dosage form" refers to physically discrete units, each containing a predetermined amount of the CD73 inhibitor, alone or in combination with one or more additional agents, sufficient to produce the desired effect. It will be appreciated that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved.

Kits

[0161]The present invention also contemplates kits comprising a CD73 inhibitor according to the first aspect, and pharmaceutical compositions thereof, and at least one additional therapeutic agent. Kits are generally in the form of a physical structure housing various components, as described below, and can be used, for example, to practice the methods described above.

[0162]A kit includes one or more of the CD73 inhibitors according to the first aspect (provided in, for example, a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. CD73 inhibitors may be supplied in a ready-to-use form (for example, a tablet or capsule) or in a form that requires, for example, reconstitution or dilution (for example, a powder) before administration. When the CD73 inhibitors are in a form that needs to be reconstituted or diluted by a user, the kit may also include diluents (e.g., sterile water), buffers, pharmaceutically acceptable excipients, and the like, packaged with or separately from the inhibitors. from<c>D73. The kit may contain the different agents separately or they may be already combined in the kit. Each component of the kit may be within an individual package, and all of the various packages may be within a single package. A kit of the present invention may be designed for the conditions necessary to adequately maintain the components housed therein (e.g., refrigeration or freezing).

[0163] A kit may contain a label or package insert that includes identifying information of the components it contains and instructions for their use (e.g., dosing parameters, clinical pharmacology of the active ingredient or ingredients, including the mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). Labels or inserts may include manufacturer information such as lot numbers and expiration dates. The label or packaging insert may be, e.g. e.g., integrated into the physical structure that houses the components, contained separately within the physical structure, or attached to a component of the kit (e.g., an ampoule, a tube, or a vial).

e x p e r im e n t a l

[0164] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent that the inventors experiments set out below have been carried out or are all the experiments that can be carried out. It should be understood that exemplary descriptions written in the present tense were not necessarily made, but rather the descriptions may be made to generate data and the like of the nature described therein. Efforts have been made to ensure the accuracy of the figures used (e.g. quantities, temperature, etc.), but some experimental errors and deviations must be taken into account.

[0165] Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius (°C), and pressure is atmospheric or near atmospheric. Standard abbreviations are used, including the following: wt = wild type; bp = base pair(s); kb = kilobase(s); nt = nucleotide(s); aa = amino acid(s); s or sec = second(s); min = minute(s); h or hr = hour(s); ng = nanogram; |jg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; j l or j L = microliter; ml or mL = milliliter; 1 or L = liter; jM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. = intramuscular(ly); i.p. i.p. = intraperitoneal; SC or SQ = subcutaneous; QD = journal; BID = twice a day; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; IHC = immunohistochemistry; DMEM = Dulbeco modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0166] LC: Agilent 1100 series; Mass spectrometer: Agilent G6120BA, single quad LC-MS method: Agilent Zorbax Eclipse PI<us>C 18.4.6 x<i>o 0 mm, 3.5 jM , 35 °C, 1.5 mL flow rate /min, 2.5 min gradient from 0% to 100% B with 0.5 min wash to 100% B; A = 0.1% formic acid / 5% acetonitrile / 94.9% water; B = 0.1% formic acid / 5% water / 94.9% acetonitrile.

Flash Column: ISCO Rf+

Reversed phase HPLC: ISCO-EZ; Column: Kinetex 5 jm EVO C18 100 A; 250 x 21.2mm (Phenomenex)

Reference Example 1

Synthesis of [({[(2R,3S,4R,5R))-5-[6-(cidopentylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy acid }(hydroxy)phosphoryl)methyl]phosphonic

[0167]

[0168] Step a: A mixture of 2,6-didoropurine riboside (321 mg, 1 mmol), cydopentylamine (103 pl, 1.05 mmol, 1.05 equiv.) and triethylamine (146 pl, 1.05 mmol , 1.05 equiv.) in anhydrous EtOH (3 mL) was stirred at 60 0C overnight. The reaction mixture was evaporated and the crude product was used in the next step without purification. ESI MS [M+H]+ for C15H21CIN5O4, cal. 370.8, found 370.2

[0169] Step b: The product from step a (370 mg, 1 mmol) was dissolved in trimethylphosphate (5 mL) and cooled to 0 ° C (ice bath), then a cold solution of methylenbis was added dropwise (phosphonide diioride) (1.25 g, 5 mmol, 5 equiv.) in trimethylphosphate (2 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (7 mL) and stirred at 0 °C for 15 min, and then 2 h at room temperature. . The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 30% aeetonitrile and water with 0.1% TFA) to give the product as a white solid in 28% yield (181 mg). : 1H NMR (400 MH<z>, DMSO) 68.45 - 8.32 (m, 2H), 5.85 (d,J= 5.5 H<z>, 1H), 4.55 - 4.36 (m, 2H), 4.23 - 4.07 (m , 4H), 2.26 (t,J= 20.5 H<z>, 2H), 2.04 - 1.85 (m, 2H), 1.77 - 1.46 (m, 6H). ESI MS [M+H]+ for C16H25CIN5O9P2, cal. 528.8, found 528.1

Reference Example 2

Synthesis of acid (((((2R,3S,4R,5R))-5-(6-((4-(ert-butyl)benzyl)amino)-2-chloro-9H-purin-9-yl}-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0170]

[0171]The base compound was synthesized in a similar manner to Example 1 using 4-tert-butylbeneylamine instead of eylopentylamine: 1H NMR (400 MHz, DMSO-c6) 68.91 (t,J= 6.3 Hz, 1H), 8.43 (s , 1H), 7.33 (d,J= 8.2 Hz , 2H), 7.26 (d,J= 8.2 Hz , 2H), 5.86 (d,J= 5.8 Hz , 1H), 4.68 - 4.56 (m, 2H), 4.52 (t,J= 5.4 Hz, 1H), 4.23 - 4.03 (m, 4H), 2.26 (t,J= 20.5 Hz, 2H), 1.25 (s, 9H). ESI MS [M+H]+ for C22H31CIN5O9P2, cal. 606.1, found 606.2.

Reference Example 3

Synthesis of acid (((((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3,4 dihydroxytetrahydrofuran-2-yl)methoxy) (hydroxy)1bsphoryl)methyl)1bsphonic

[0172]

[0173]The base compound was synthesized in a similar way to Example 1 using isopropylamine instead of eielopentylamine: <1>H NMR (400 MHz, DMSO-d<6>) 6 8.40 (s, 1H), 8.23 (d,J= 8.1 Hz , 1H), 5.85 (d,J= 5.9 Hz , 1H), 4.51 (t,J= 5.5 Hz , 1H), 4.36 (s, 1H), 4.24 - 4.03 (m , 4H), 2.25 (t,J = 20.5 Hz, 2H), 1.21 (dd,J= 6.6, 2.0 Hz, 5H). ESI MS [M+H]+ for C14H22CIN5O9P2, cal. 502.1, found 502.1

Reference Example 4

Synthesis of (((((2R,3S,4R,5R))-5-(2-chloro-6-(cyclopropylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy acid )(hydroxy)phosphoryl)methyl)phosphonic

[0174]

[0175]The base compound was synthesized similarly to Example 1 using cidopropylamine instead of cidopentylamine: 1H NMR (400 MH<z>, DMSO-deJ 68.54 (<s>, 1H), 8.42 (<s>, 1H), 5.86 (d,J= 5.8 H<z>, 1H), 4.52 (t,J= 5.4 H<z>, 1H), 4.28 - 4.03 (m , 4H), 2.97 (s, 1H), 2.25 (t,J = 20.5 Hz , 2H), 0.75 (s, 2H), 0.64 (s, 3H). ESI MS [M+H]+for C14H20CIN5O9P2, cal.

Reference Example 5

Synthesis of (((((2R,3S,4R,5R))-5-(2-chloro-6-(neopentylamino}-9H-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-yl)methoxy acid synthesis )(hydroxy)phosphoryl)methyl)phosphonic

[0176]

[0177]The base compound was synthesized similarly to Example 1 using neopentylamine instead of cidopentylamine: 1H NMR (400 MH<z>, DMSO-deJ 68.42 (<s>, 1H), 8.32 (t,J= 6.4 H<z >, 1H), 5.85 (d,J= 5.7 H<z>, 1H), 4.52 (t,J= 5.4 H<z>, 1H), 4.31 - 4.04 (<m>, 4H), 3.82 (d, J= 7.0 H<z>, 1H), 3.42 - 3.17 (m, 2H), 2.26 (t,J= 20.5 H<z>, 2H), 0.91 (<s>, 9H).ESI MS [M+H ]+ for C16H26CIN5O9P2, cal. 530.1, found 530.2

Reference Example 6

Synthesis of acid (((((2R,3S,4R,5R))-5-(2-chloro-6-(isopropyl(methyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0178]

[0179]The base compound was synthesized similarly to Example 1 using N-methylisopropylamine instead of cidopentylamine: 1H NMR (400 MH<z>, DMSO-d 6) 68.42 (<s>, 1H), 5.88 (d,J= 5.9 H<z>, 1H), 4.50 (t,J= 5.4 H<z>, 1H), 4.22 -4.17 (m, 1H), 4.11 (d,J= 6.4 H<z>, 3H), 3.03 ( <s>, 3H), 2.26 (t,J= 20.5 H<z>, 2H), 1.23 (<s>, 6H). ESI MS [M+H]+for C15H24CIN5O9P2, cal. 516.1, found 516.1

Reference Example 7

Synthesis of (((((2R,3S,4R,5K)-5-(6-((3,5-bis(trifluoromethyl)benzyl)aminopurin-9-yl)-3,4-dihydroxytetrahydrofuran-2yl)methoxy acid )(hydroxy)phosphoryl)methyl)phosphonic

[0180]

[0181] The base compound was synthesized in a similar way to Example 1, but using 6-eIoropurine riboside and 3,5-bis(trifIuorometiI)beneiIamine in the step a:<1>H NMR (400 MH<z>, DMSO-deJ 68.67 (<s>, 1H), 8.47 (<s>, 1H), 8.26 (<s>, 1H), 8.07 (s, 2H), 7.99 (s, 1H), 5.94 (d,J= 5.7 Hz, 1H), 4.88 (s, 2H), 4.61 (t,J= 5.4 Hz, 1H), 4.23 (t,J= 4.2 Hz, 1H), 4.20 - 4.04 (m, 3H), 2.25 (t,J= 20.5 Hz , 2H). ESI MS [M-H]- for C20H20F6N5O9P2, cal.

Reference Example 8

Synthesis of (((((2RJ3SJ4RJ5R)-5-(6-((4-bromobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl acid )methyl)phosphonic

[0182]

[0183]The base compound was synthesized in a similar way to Example 1, but using 6-eIoropurine riboside and the corresponding amine in the step a: <1>H NMR (400 MHz, DMSO-d<6>) 68.60 (s, 1H ), 8.44 (s, 1H), 8.24 (s, 1H), 7.49 (d,J= 8.3 Hz , 2H), 7.29 (d,J= 8.3 Hz , 2H), 5.94 (d,J= 5.7 Hz , 1H ), 4.67 (s, 2H), 4.61 (t,J= 5.3 Hz , 1H), 4.23 (t,J= 4.2 Hz , 1H), 4.19 - 4.05 (m , 3H), 2.25 (t,J= 20.5 Hz , 2H). ESI MS [M-H]- for C<1>sH<21>BrN<5>0 gP<2>, cale. 592.0, found 592.1.

Reference Example 9

Synthesis of (((((2RJ3SJ4RJ5R)-5-(6-((4-(ert-butyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy acid )(hydroxy)phosphoryl)methyl)phosphonic

[0184]

[0185]The base compound was synthesized similarly to Example 1, but using 6-eIoropurine riboside and the corresponding amine in the a step: 1H NMR (400 MH<z>, DMSO-d6) 68.62 (<s>, 1H) , 8.45 (<s>, 1H), 8.26 (<s>, 1H), 7.37 -7.22 (m, 4H), 5.94 (d,J= 5.7 H<z>, 1H), 4.67 (<s>, 2H ), 4.60 (t,J= 5.4 H<z>, 1H), 4.23 (t,J= 4.1 H<z>, 1H), 4.20 -4.05 (<m>, 3H), 2.25 (t,J= 20.5 Hz, 2H), 1.24 (s, 9H). ESI MS [M - ^ for C22H30N5O9P2, cal. 570.1, found 570.3.

Reference Example 10

Synthesis of acid (((((2Rí3Si4Rí5R)-S-(6-(([1,1,-biphenyl]-4-ylmethyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0186]

[0187]The base compound was synthesized similarly to Example 1, but using 6-eloropurine riboside and the corresponding amine in the a step: H NMR (400 MHz, DMSO-d<6>) 68.67 (s, 1H) , 8.46 (s, 1H), 8.27 (s, 1H), 7.66 -7.57 (m, 4H), 7.49 - 7.40 (m, 4H), 7.37 - 7 .30 (m, 1H), 5.95 (d, J = 5.7 H<z>, 1H), 4.76 (<s>, 2H), 4.61 (t, J = 5.3 H <z>, 1H), 4.24 (t, J = 5.3 H<z>, 1H).95 (d,J= 5.7 H<z>, 1H), 4.76 (<s> , 2H), 4.61 (t,J= 5.3 H<z>, 1H), 4.24 (t,J= 4.1 H<z>, 1H), 4.20 - 4.06 ( m, 3H), 2.25 (t,J=20.5 Hz, 2h). e S i MS [M-H]" for C24H26N5O9P2, cal. 590.1, found 590.2.

Reference Example 11 Synthesis of acid (((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((4-(trifluoromethyl)benzyl)amino)-9H-purin-9- il)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic.

[0188]

[0189]The base compound was synthesized similarly to Example 1, but using 6-eloropurine riboside and the corresponding amine in the step a:<1>H NMR (400 MHz, DMSO-d<6>) 68.68 (s, 1H), 8.46 (s, 1H), 8.25 (s, 1H), 7.67 (d,J= 8.1 Hz, 2H), 7.54 (d,J= 8.1 Hz, 2H), 5.95 (d,J= 5.8 Hz, 1H), 4.79 (s, 2H), 4.61 (t,J= 5.3 Hz, 1H), 4.24 (t,J= 4.1 Hz, 1H), 4.20 -4.06 (m, 3H), 2.25 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C19H21F3N5O9P2, cal. 582.1, found 582.2.

Reference Example 12

Synthesis of (((((2R,3S,4R,5R))-3,4-dihydroxy-5-(6-((4-methylbenzyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl acid )methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0190]

[0191]The base compound was synthesized similarly to Example 1, but using 6-eloropurine riboside and the corresponding amine in the a step: 1H NMR (400 MH<z>, DMSO-d6) 68.61 (<s>, 1H) , 8.45 (<s>, 1H), 8.26 (<s>, 1H), 7.22 (d,J= 7.8 H<z>, 2H), 7.10 (d,J= 7.8 H<z>, 2H), 5.94 (d,J= 5.7 H<z>, 1H), 4.67 (<s>, 2H), 4.60 (t,J= 5.4 H<z>, 1H), 4.23 (t,J= 4.2 H<z>, 1H), 4.19 -4.04 (m , 3H), 2.31 -2.18 (m , 5H). ESI MS [M-H]- for C19H24N5O9P2, cal. 528.1, found 528.2.

Reference Example 13

Synthesis of acid (((((2R,3S,4R,5R))-5-(6-((3,5-dichlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0192]

[0193]The base compound was synthesized in a similar way to example 1, but using 6-chloropurine riboside and the corresponding amine in the step a: <1>H NMR (400 MH<z>, DMSO-d<6>) 58.61 (<s>, 1H), 8.46 (<s>, 1H), 8.26 (<s>, 1H), 7.48 (t,J= 2.0 Hz , 1H), 7.39 (s, 2H), 5.95 (d,J = 5.7 Hz , 1H), 4.70 (s, 2H), 4.61 (t,J= 5.4 Hz , 1H), 4.24 (t,J= 4.2 Hz , 1H), 4.20 - 4.05 (m , 3H), 2.26 (t ,J= 20.5 Hz , 2H). ESI MS [M-H]- for C18H20CI2N5O9P2, cal. 582.1, found 582.2.

Reference Example 14

Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-methyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy acid )(hydroxy)phosphoryl)methyl)phosphonic

tetraMethyltin (470 pL, 3.34 m m o I) in NMP (10 m L) Pd(PPh3)4 (196 Mg, 0.17 m m o I, 10 m m o I%) was added and the reaction mixture was heated to 120 ° C for one night. The LCMS indicated the product form. It was cooled to room temperature, diluted with water, extracted with ethyl acetate, dried (MgSO4), filtered and concentrated. The residue was purified by flash light to obtain the product (1 g). ESI MS [M+H]+ for C24H27N5O7, cal. 498.2, found 498.3

[0196] Step b: To a solution of the acetate derivative of step a (1 g, 2.01 m m o) in Methanol (5 m L) K2CO3 (276 Mg, 2 m m o I) was added and the reaction mixture was stirred at t.r. for 1 hour. It was then diluted with dieloroMethane and filtered through a silica pad. The filtrate was concentrated and purified by flash cooling (ISCO, 40 g cooling, 0 to 20% Methanol in dieloroMethane, 20 Min) to obtain the off-white solid (450 Mg, 60%). ESI MS [M+H]+ for C18H21N5O4, cal. 372.2, found 372.2

[0197] Step c: The product of step b (150 Mg, 0.4<m>o I) was dissolved in triMethylphosphate (3<m>L) and cooled to 0 ° C (ice bath), then added drop by drop an ice-cold solution of Methylenebis(phosphonium) dichloride (504 Mg, 2<m m>o I, 5 equiv.) in trimethylphosphate (1 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (8 mL) and stirred at 0 °C for 15 min, and then 2 h at room temperature. . The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from O to 30% acetonitrile and water with 0.1% TFA) to give the product as a white solid: 1H NMR (4OO MHz, DMSO-d 6) 88.48 - 8.32 (m, 2H), 7.38 - 7.18 (m, 5H), 5.92 (d,J= 6.0 H<z>, 1H), 4.71 (<s>, 2H), 4.55 (t,J= 5.5 H<z>, 1H), 4.19 - 3.98 (m, 4H), 2.44 (s, 3H), 2.23 (t,J= 2O.5 H<z>, 2H). ESI MS [M-H]- for C19H25N5O9P2, cal. 528.1, found 528.2.

Reference Example 15

Synthesis of acid (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-vinyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

(462 mg, 3 mmol, 1.5 equiv.), K2CO3 (828 mg, 6 mmol, 3 equiv.) and Pd(PPh3)4 in 1,2-dimethoxyethane:H<2>0 (9:1, 1O mL) was stirred under N2 at 85 °C for 1 day. The reaction mixture was cooled to room temperature, diluted with EtOAe (1OO mL) and washed with H2O (50 mL). The organic layer was separated, dried over MgS0<4>, filtered and evaporated to give a yellow solid. The crude product was washed with MTBE (50 mL) and used directly in the next step (550 mg, 72%).

[0200] Step b: The base compound was synthesized in a similar manner to example 1. <1>H NMR (4OO MHz, DMSO-d<6>) 88.39 (s, 1H), 7.38 (d,J= 7.0 H< z>, 2H), 7.29 (t,J= 7.6 H<z>, 2H), 7.25 - 7.15 (m, 1H), 6.64 (dd,J= 17.2, 1O.4 H<z>, 1H), 6.39 (dd,J= 17.2, 2.4 Hz, 1H), 5.94 (d,J= 6.O Hz, 1H), 5.55 (d,J= 1O.5 Hz, 1H), 4.73 (s, 2H), 4.63 ( t,J= 5.5 Hz , 1H), 4.28 - 4.OO (m, 4H), 2.25 (t,J= 2O.4 Hz , 2H). ESI MS [M+H]+ for C20H26N5O9P2, cal. 542.1, found 542.2.

[0201] Step c: The product from step b (40 mg, 0.06 mmol) was dissolved in MeOH (10 mL), purged with N2 and 10% Pd/C (50% wet, 30 mg) was added ). The reaction mixture was stirred vigorously under h2 (balloon) for 2h and after filtration the product was purified by HPLC RP18 (H2O+0.1o/o TFA/aeetonitrile+O.1% TFA) to give white solid (14 mg, 35%):<1>H NMR (4OO MH<z>, DMSO-d<6>) 88.52 - 8.18 (m, 2H), 7.33 - 7.27 (m, 2H), 7.27 - 7.18 (m, 2H), 7.15 (t,J= 7.2 H<z>, 1H), 5.86 (d,J= 6.O Hz , 1H), 4.64 (s, 2H), 4.55 (t,J= 5.5 Hz , 1H), 4.19 - 3.98 (m, 4H), 2.7O - 2.61 (m, 2H), 2.16 (t,J= 2O.5 Hz, 2H), 1.16 (t,J= 7.6 Hz, 3H). ESI MS [M+H]+ for C20H27N5O9P2, cal. 544.1, found 544.2.

Reference Example 16

Synthesis of acid (((((2R,3S,4R,5R)-5-(2-allyl-6-(benzylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0202]

[0203]The base compound was synthesized in a similar manner to example 15.<1>H NMR (400 MHz, DMSO-de) 6 8.46 (s, 1H), 8.37 (s, 1H), 7.41 - 7.34 (m, 2H ), 7.30 (t,J= 7.5 Hz, 2H), 7.25 -7.18 (m, 1H), 6.17 - 6.03 (m , 1H), 5.92 (d,J= 6.0 Hz , 1H), 5.28 - 5.00 (<m >, 2H), 4.70 (<s>, 2H), 4.60 (t,J= 5.6 Hz, 1H), 4.27 - 4.02 (<m>, 4H), 3.49 (d,J= 6.8 Hz, 2H), 2.24 (t,J= 20.5 H<z>, 2H). ESI m S [M+H]+ for C21H28N5O9P2, cal. 556.1, found 556.3.

Reference Example 17

Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-propyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy acid )(hydroxy)phosphoryl)methyl)phosphonic

[0204]

[0205] The base compound was synthesized similarly to the step of Example 15: <1>H NMR (400 MHz, DMSO-d e) 6 8.40 (<s>, 2H), 8.29 (<s>, 1H), 7.29 (d,J= 7.6 Hz, 2H), 7.23 (t,J= 7.5 Hz, 2H), 7.15 (t,J= 7.2 Hz, 1H), 5.86 (d,J= 6.0 H<z>, 1H), 4.64 (s, 2H), 4.54 (t,J= 5.5 Hz, 1H), 4.19 - 3.94 (m, 4H), 2.71 - 2.55 (m, 2H), 2.17 (t,J= 20.5 Hz, 2H), 1.66 (q,J= 7.4 Hz, 2H), 0.93 - 0.70 (m , 3H). ESI MS [M+H]+ for C21H30N5O9P2, cal. 558.1, found 558.2.

Reference Example 18

Synthesis of [({[(2R,3S,4R,5R))-5-[6-(benzylamino)-2-methoxy-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy acid }(hydroxy)phosphoryl)methyl]phosphonic

[0206]

[0207] Step a: The known riboside (250 mg, 0.64 mmol) was dissolved in 25% NaOMe solution in MeOH (2 mL) and stirred at 60 ° C overnight. The reaction mixture was concentrated under reduced pressure and the residue was diluted with H2O (15 mL) and acetic acid to neutral pH. The product was collected by filtration (white solid, 180 mg, 73%). ESI MS [M+H]+ for C18H22N5O5, cal. 388.4, found 388.1.

[0208] Step b: The base compound was obtained by a procedure similar to that of example 1 to give a white solid (37 mg, 14%): 1H NMR (400 MH<z>, DMSO) 68.48 (<s>, 1H ), 8.20 (<s>, 1H), 7.37 -7.17 (m, 5H), 5.82 (d,J= 5.9 H<z>, 1H), 4.64 (d,J= 5.0 H<z>, 3H), 4.28 - 4.00 (m, 4H), 3.80 (<s>, 3H), 2.23 (t,J= 20.5 H<z>, 2H). ESI MS [M+H]+ for C19H26N5O10P2, cal. 546.4, found 546.1.

Reference Example 19

Synthesis of [({[(2R,3S,4R,5R))-5-[6-(benzylamino)-2-(methylamino)-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl acid ]m ethoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0209]

[0210] Step a: The known riboside (250 mg, 0.64 mmol) was dissolved in 40% MeNH2 solution in H2O (2 mL) and stirred at 60 ° C overnight. The reaction mixture was then concentrated under reduced pressure and the residue was diluted with H2O (15 mL). The product was collected by filtration (white solid, 210 mg, 85%). ESI MS [M+H]+for C18H23N6O4, cal. 387.4, found 387.3.

[0211] Step b: The base compound was obtained by a procedure similar to that of Example 1 to give a white solid (38 mg, 15%): 1H NMR (400 MH<z>, DMSO) 68.08 (<s>, 1H ), 7.42 -7.19 (m, 5H), 5.79 (d,J= 6.1 H<z>, 1H), 4.75 -4.45 (m, 3H), 4.24 - 4.02 (m, 4H), 2.81 (<s>, 3H), 2.22 (t,J= 20.4 H<z>, 2H). ESI MS [M-H]- for C19H26N6O9P2, cal. 543.4, found 543.2.

Reference Example 20

Synthesis of (((((2R,3S,4R,5R))-5-(6-(benzylamino)-2-(dimethylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0212]

[0213] The base compound was synthesized in a similar manner to Example 19 but using dimethylamine in step a: <1>H NMR (400 MHz, DMSO-d a) 68.15 (s, 1H), 8.09 (s, 1H), 7.36 (d,J= 7.2 Hz, 2H), 7.29 (t,J= 7.5 Hz, 2H), 7.21 (t,J= 7.2 Hz, 1H), 5.81 (d,J= 5.5 Hz, 1H), 4.68 - 4.57 (m, 3H), 4.26 - 4.20 (<m>, 1H), 4.20 - 4.00 (<m>, 3H), 3.06 (<s>, aH), 2.24 (t,J= 20.4 Hz, 2H). ESI MS [M+H]+ for C<2>oH<29>Na0<9>P<2>, cal. 559.1, found 559.2.

Reference Example 21

Synthesis of acid (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(pyrrolidin-1-yl)-9H-purin-9-yl}-3,4-dihydroxytetrahydro1furan -2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0214]

[0215] The base compound was synthesized in a similar way to Example 19 but using pyrrolidine in step a: <1>H NMR (400 MHz, DMSO-d a) 68.15 (s, 2H), 7.42 -7.14 (m, 5H ), 5.82 (d,J= 5.5 Hz, 1H), 4.71 -4.51 (m, 3H), 4.26 (t,J= 4.3 Hz, 1H), 4.21 - 4.00 (m, 3H), 3.46 (s, 4H) , 2.23 (t,J= 20.4 Hz , 2H), 1.89 (s, 4H). ESI MS [M+H]+ for C<22>Hs<1>Na0<9>P<2>, cal.

585.1, found 585.2.

Reference Example 22

Synthesis of acid (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(piperidin-1-yl)-9H-purin-9-yl)-3,4-dihydroxytetrahydro1furan -2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0216]

[0217] The base compound was synthesized in a similar way to Example 19 but using piperidine in step a: <1>H NMR (400 MHz, DMSO-d a) 68.20 (s, 1H), 8.10 (s, 1H), 7.38 -7.33 (m, 2H), 7.33 -7.25 (m, 2H), 7.25 -7.16 (m, 1H), 5.81 (d,J= 5.a Hz, 1H), 4.aa -4.52 (m, 3H ), 4.20 (t,J= 4.3 Hz, 1H), 4.17 -4.00 (m , 3H), 3.74 -3.62 (m, 4H), 2.24 (t,J= 20.5 Hz , 2H), 1.64 - 1.38 (m, aH). ESI MS [M-H]- for C<2>sH<31>Na0<9>P<2>, cal. 597.2, found 597.3.

Reference Example 23

Synthesis of acid (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-morpholino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2yl)m ethoxy) (hydroxy)phosphoryl)methyl)phosphonic

[0218]

[0219] The base compound was synthesized in a similar way to Example 19 but using morpholine in step a:<1>H NMR (400 MH<z>, DMSO-d<6>) 68.24 -8.02 (<m>, 2H ), 7.37 -7.17 (<m>, 5H), 5.79 (d,J= 5.9 Hz, 1H), 4.72 -4.51 (<m>, 3H), 4.23 -3.99 (m, 4H), 3.61 (s, 8H ), 2.23 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C22H29N6O10P2, cal. 599.2, found 599.3

Reference Example 24

Synthesis of (((((2R,3S,4R,5R))-5-(6-(benzylamino)-2-(isopropylthio)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)m ethyl)phosphonic

[0220]

[0221] Step a: A solution of compound The mixture was then warmed to room temperature and stirred until the reaction was complete. Nitrogen was then bubbled through the reaction to remove excess ammonia gas. The mixture was concentrated and purified by prep HPLC. to give the desired product (750 mg, 20%).

[0222] Step b: The product of step a (0.36 g, 1 mmol), beneyl amine (0.115 mL, 1.05 mmol, 1.05 equiv.) and Et3N (0.15 mL, 1.1 mmol, 1.1 equiv.) in anhydrous EtOH (3.3 mL) was stirred at 70 °C for 4 hours. The reaction mixture was then cooled to room temperature, concentrated and used without further purification.

[0223] Step c: The product from step b was dissolved in trimethylphosphate (5 mL) and cooled to 0 ° C (ice bath), then a cold solution of methylenebis (phosphonic dieloride) was added dropwise (1 .2 g, 15 mmol, 5 equiv.) in trimethylphosphate (3 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (6 mL) and stirred at 0 °C for 15 min, and then 2 h at room temperature. . The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 40% aeetonitrile and water with 0.1% TFA) to give the product as a white solid in a yield of 6% (38 mg). : 1H NMR (400 MHz, DMSO-d 6) 6 8.53 (<s>, 1H), 8.27 (<s>, 1H), 7.37 -7.17 (m, 5H), 5.84 (d,J= 5.8 Hz, 1H ), 4.65 (<s>, 2H), 4.56 (t,J= 5.5 Hz, 1H), 4.24 -4.17 (m, 1H), 4.17 -4.01 (m, 3H), 3.82 -3.71 (m, 1H), 2.24 (t,J= 20.5 Hz, 2H), 1.28 (d,J=6.8 Hz, 6H).. ESI MS [M+H]+for C21H29N5O9P2S, cale. 590.1, found 590.2

Reference Example 25

Synthesis of (((((2R,3S,4R,5R))-5-(6-(benzylamino}-2-(isopropylsul1onyl)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0224]

[0225] Step a: The product from step a of Example 24 (4.5 g, 12.5 mmol) in methylene chloride (50 mL) was treated with m-CPBA (2.2 g, 38.2 mmol ) by portions. The reaction was stirred at room temperature until complete. The mixture was diluted with methylene chloride (200 mL), washed with aqueous NaHS0<3>twice, dried over Na<2>S0<4>and concentrated. The residue was purified by prep HPLC. to give the desired product as a white solid (780 mg, 16%).

[0226] Step b: The product of step a (0.393 g, 1 mmol), benzyl amine (0.115 mL, 1.05 mmol, 1.05 equiv.) and Et3N (0.15 mL, 1.1 mmol, 1.1 equiv.) in anhydrous EtOH (3.3 mL) was stirred at 70 °C for 4 hours. The reaction mixture was then cooled to room temperature, concentrated and used without further purification.

[0227] Step c: The product from step b was dissolved in trimethylphosphate (4 mL) and cooled to 0 ° C (ice bath), then a cold solution of methylenebis (phosphonic dichloride) (1) was added dropwise .2 g, 5 mmol, 5 equiv.) in trimethylphosphate (2 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (6 mL) and stirred at 0 °C for 15 min, and then 2 h at room temperature. . The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 40% acetonitrile and water with 0.1% TFA) to give the product as a white solid in a yield of 22% (50 mg). :<1>H NMR (400 MHz, DMSO-d<6>) 69.21 (t,J=6.2 Hz, 1H), 8.66 (s, 1H), 7.42 -7.15 (m, 5H), 5.97 (d,J = 6.1 Hz, 1H), 4.74 -4.66 (m, 2H), 4.60 (dd,J= 6.1,5.0 Hz , 1H), 4.26 -4.22 (m, 1H), 4.19 -4.07 (m, 4H), 3.78 ( p,J= 6.8 Hz, 1H), 2.26 (t,J= 20.5 H<z>, 2H), 1.12 (dd,J= 6.8, 2.4 Hz, 6H). ESI MS [M+H]+ for C21H29N5O 11P2S, cal. 622.1, found 622.2

Reference Example 26

Synthesis of acid (((((2R,3S,4S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)m ethoxy) (h idroxy)phosphoryl)m ethyl)phosphonic

[0228]

[0229] The base compound was synthesized similarly to step b of Example 1 using the corresponding alcohol: <1>H NMR (400 MHz, DMSO-d<6>) 68.17 (s, 1H), 8.02 -7.72 ( m, 2H), 6.15 (d,J= 4.3 Hz, 1H), 4.30 -4.09 (m, 4H), 4.00 - 3.88 (m, 1H), 2.24 (t,J=20.5 Hz, 2H). ESI MS [M+H]+ for C11H17FN5O9P2, cal. 444.0, found 444.1.

Reference Example 27

Synthesis of acid (((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl}-4-fluoro-3-hydroxytetrahydrofuran-2-yl)m ethoxy)(h idroxy)phosphoryl)m ethyl)phosphonic

[0230]

[0231] The base compound was synthesized similarly to step b of Example 1 using the corresponding alcohol: 1H NMR (400 MH<z>, DMSO-da) 68.45 (<s>, 1H), 8.26 (<s> , 1H), 7.92 (<s>, 2H), 6.27 (dd,J= 17.2, 2.8 Hz, 1H), 5.50 (ddd,J= 52.5, 4.5, 2.8 Hz, 1H), 4.64 -4.52 (m, 1H ), 4.29 -4.08 (<m>, 3H), 2.25 (t,J= 20.4 H<z>, 2H). ESI MS [M+H]+ for C11H16FN5O<s>P2, cal. 428.1, found 428.1.

Reference Example 28

Synthesis of [({[(2R,3R,4S,5R}-5-(6-amino-2-chloro-9H-purin-9-yl}-4-fluoro-3-hydroxyoxolan-2-yl] methoxy acid) } (hydroxy}phosphoryl}m ethyl]phosphonic

[0232]

[0233] The base compound was synthesized similarly to Example 1 using commercially available alcohol. 1H NMR (400 MHz, DMSO-d a) 68.28 (d,J= 2.2 Hz, 1H), 7.92 (s, 2H), 6.36 (dd,J= 14.3, 4.6 Hz, 1H), 5.26 (dt,J= 52.5, 4.3 Hz, 1H), 4.51 (dt,J= 18.6, 4.7 Hz, 1H), 4.19 (t,J= 6.0 Hz, 2H), 4.04 (t,J=5.0 Hz, 1H), 2.26 (t, J= 20.5 Hz, 2H); MS: (ES) m/z calculated for C11H15CIFN5O8P2 [M-H]- 46o.1, found 460.1.

Reference Example 29

Synthesis of acid (((((2R,3R,4S,5R}-5-(6-(benzylamino}-2-chloro-9H-purin-9-yl}-4-fluoro-3-hydroxytetrahydrofuran-2-yl }m ethoxy}(h idroxy}phosphoryl}m ethyl}phosphonic

[0234]

[0235] Step a: 2,6-didoropurine (3.6 g, 18.8 mmol) was dissolved in 90 mL of acetonitrile and treated with CS2CO3 (7.5 g, 23 mmol, 1.2 equiv.) . The mixture was stirred at room temperature for 30 min. The known brominated derivative (8.75 g, 21 mmol, 1.1 equiv.) was dissolved in 100 mL of acetonitrile and added to the mixture dropwise through an addition funnel. The mixture was allowed to stir overnight at room temperature. The mixture was filtered onto a silica gel pad and concentrated. The residue was adsorbed onto silica and purified by column chromatography (hexanes/ethyl acetate) to obtain the product as a white solid in 77% yield (7.72 g). 1H NMR (400 MHz, Chloroform-d) 68.39 (d,J= 3.0 Hz, 1H), 8.10 (ddt,J= 8.5, 3.1, 0.9 Hz, 4H), 7.74 -7.36 (m, 6H), 6.64 (dd ,J= 21.8, 2.8 Hz, 1H), 5.83 - 5.69 (m, 1H), 5.40 (ddd,J= 49.9, 2.8, 0.8 Hz, 1H), 4.89 - 4.77 (m, 2H), 4.62 (q,J = 4.0 H<z>, 1H). ESI MS [M+H]+ for C24H17CI2FN4O5, cal. 531.1, found 531.1.

[0236] Step b: The product of step a (9.0 g, 17 mmol), beneyl amine (3 mL, 26 mmol, 1.5 equiv.) and EtsN (5 mL, 34 mmol, 2.0 equiv. .) in anhydrous EtOH (60 mL) was stirred at 70 °C for 4 hours. The reaction mixture was then cooled to room temperature and the product was collected by filtration and used without further purification (white solid, 8.9 g, 87%). ESI MS [M+H]+ for C31H25CIFN5O5, cal. 602.2, found 602.0.

[0237] The above product (10.2 g, 17 mmol) and <k>2<c>o3 (7 g, 51 mmol, 3 equiv) were dissolved in 170 mL of methanol and stirred at room temperature for 4 hours . The reaction mixture was then filtered and concentrated on a silica gel pad. The reaction mixture was purified by column chromatography (methylene chloride/methanol) to obtain the product as a white solid in 80% yield (5.3 g): 1H NMR (400 MHz, DMSO-d6) 68.97 ( t,J= 6.3 Hz, 1H), 8.31 (d,J= 2.0 Hz, 1H), 7.36 - 7.18 (m, 5H), 6.34 (dd,J= 13.6, 4.7 Hz, 1H), 5.23 (dt,J = 52.6, 4.3 Hz, 1H), 4.66 (q,J= 7.3, 5.7 Hz, 2H), 4.43 (dt,J= 19.0, 4.8 Hz, 1H), 3.84 (q,J= 4.9 Hz, 1H), 3.65 (tq,J= 12.0, 6.2, 5.2 Hz, 2H). ESI MS [M+H]+ for C17H18CIFN5O3, cal. 394.1, found 394.1.

[0238] Step <c>: The product of step b (800 mg, 2 mmol) was dissolved in trimethylphosphate (15 mL) and cooled to 0 ° C (ice bath), then a solution was added dropwise cold methienbis(phosphonium dieIoride) (2.5 g, 10 mmol, 5 equiv.) in trimethylphosphate (5 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (15 mL) and stirred at 0 °C for 15 min, and then 2 h at room temperature. . The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 40% acetonitrile and water with 0.1% TFA) to give the product as a white solid in a yield of 22% (290 mg). : 1H NMR (400 MHz, DMSO-d6) 68.99 (t,J= 6.3 Hz, 1H), 8.30 (d,J= 2.2 Hz, 1H), 7.40 -7.18 (m, 5H), 6.38 (dd,J= 14.3, 4.6 Hz, 1H), 5.45 -5.04 (m, 1H), 4.65 (t,J= 5.5 Hz, 2H), 4.54 -4.42 (m, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.04 (t, J= 5.1 Hz, 1H), 2.26 (t,J= 20.5 H<z>, 2H). ESI MS [M-H]- for C18H21CIFN5O8P2, cal. 550.8, found 550.2.

Reference Example 30

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzyl(methyl)amino)-purin-9-yl}-4-fluoro-3-hydroxytetrahydrofuran-2-yl)m ethoxy)(h idroxy)phosphoryl)m ethyl)phosphonic

[0239]

[0240] The base compound was synthesized in a similar way to example 29 using the product of step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d 6) as a mixture of rotamers 68.32 (d,J= 2.1 Hz, 1H), 7.40 -7.19 (m, 5H), 6.42 (dd,J= 14.5, 4.6 Hz, 1H), 5.55 (s, 1H), 5.27 (dt,J= 52.4, 4.2 Hz, 1H), 4.95 (s, 1H), 4.50 (dt,J= 18.4, 4.5 Hz, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.05 (q,J= 5.0 Hz, 1H), 3.65 (s, 1H ), 3.11 (s, 2H), 2.26 (t,J= 20.5 Hz , 2H).ESI MS [M+H]+ for C19H23CIFN5O8P2, cal. 566.1, found 566.2

Reference Example 31a

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )m ethoxy)(hydroxy)phosphoryl)m ethyl)phosphonic

[0241]

[0242] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MH<z>, DMSO-d a) 6 8.36 (q,J= 4.6 H<z>, 1H), 8.27 (<s>, 1H), 6.45 (brs, 2H), 6.37 (dd,J= 14.3, 4.6 H<z>, 1H), 5.25 (dt,J= 52.4, 4.3 H<z>, 1H), 4.50 (dt,J= 18.6, 4.6 H<z>, 1H), 4.19 (t,J= 5.9 H<z>, 2H), 4.04 (q,J= 5.2 Hz, 1H), 3.33 (brs, 1H), 2.93 (d,J= 4.5 Hz, 3H), 2.26 (t,J= 20.4 Hz, 2H). ESI MS [M-H]- for C12H17CIFN5O8P2, cal. 474.7, found 474.1.

Reference Example 31b

Synthesis of (((((2R,3R,4S,5R))-5-(2-chloro-6-(ethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(h idroxy)phosphoryl)m ethyl)phosphonic

[0243]

[0244] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 6 8.43 (t,J= 5.7 Hz, 1H), 8.26 (s, 1H), 7.28 (brs, 2H), 6.37 (dd,J= 14.3, 4.6 Hz, 1H), 5.25 (dt,J= 52.4, 4.3 Hz, 1H), 4.50 (dt,J = 18.5, 4.6 Hz, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.03 (q,J= 5.1 Hz, 1H), 3.87 (brs, 1H), 3.45 (m, 1H), 2.27 (t ,J= 20.5 Hz , 2H), 1.17 (t,J= 7.2 Hz , 3H). ESI MS [M+H]+for C13H19CIFN5O8P2, cal. 490.7, found 490.1.

Reference Example 32

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0245]

[0246] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 68.27 (m, 2H), 6.37 (d ,J= 13.9 Hz , 1H), 5.28 (brs, 2H), 5.25 (d,J= 52.1 H<z>, 1H), 4.98 (brs, 1H), 4.51 (d,J= 18.3 H<z>, 1H), 4.35 (sept,J= 7.9 H<z>, 1H), 4.19 (m, 2H), 4.04 (m, 1H), 2.26 (t,J= 20 H<z>, 2H), 1.21 (dd ,J= 6.6, 2.1 H<z>, aH). ESI MS [M-H]- for C14H21CIFN5O8P2, cal. 502.7, found 502.2.

Reference Example 33

acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2yl)m ethoxy) (h idroxy)phosphoryl)m ethyl)phosphonic

[0247]

[0248] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-da) 68.59 (s, 1H), 8.28 (d, J= 2.1 Hz, 1H), 6.38 (dd,J= 14.2, 4.6 H<z>, 1H), 5.26 (ddd,J= 52.5, 4.3, 4.3 H<z>, 1H), 4.51 (dt,J= 18.5, 4.5 H<z>, 1H), 4.19 (t,J= 6.1 H<z>, 2H), 4.03 (q,J= 5.0 H<z>, 1H), 2.98 (s, 1H), 2.36 - 2.15 (m, 2H), 0.82 - 0.48 (m, 4H). ESI MS [M-H]- for C14H18CIFN5O8P2, cal. 500.03, found 500.0.

Reference Example 34

Synthesis of acid (((((2R,3R,4S,5R))-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran- 2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonacid

[0249]

[0250] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MH<z>, DMSO-d a) 6 8.54 (<s>, 1H), 8.28 (<s>, 1H), 6.37 (dd,J= 14.2, 4.6 H<z>, 1H), 5.25 (ddd,J= 52.5, 4.3, 4.3 H<z>, 1H), 4.54 - 4.47 (m, 1H), 4.19 (t,J= 6.3 H<z>, 2H), 4.05 -4.01 (<m>, 1H), 3.81 -3.74 (m, 1H), 3.30 - 3.27 (m, 1H) , 2.26 (dd,J= 20.5, 20.5 Hz , 2H), 1.1 - 1.3 (m, 1H), 0.48 - 0.37 (m , 2H), 0.28 - 0.26 (m , 2H). ESI MS [M-H]- for C15H20C1FN5O8P2, cal. 514.1, found 514.0.

Reference Example 35

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0251]

[0252] The base compound was synthesized in a similar way to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MH<z>, DMSO-d a) 68.41 (d,J= 7.8 H<z>, 1H), 8.27 (<s>, 1H), 6.37 (dd,J= 14.4, 4.6 H<z>, 1H), 5.25 (dt,J= 52.4, 4.3 H<z>, 1H) , 4.55 - 4.37 (m, 2H), 4.19 (t,J= 6.1 H<z>, 2H), 4.03 (q,J= 5.1 H<z>, 1H), 2.26 (t,J= 20.5 H<z >, 2H), 1.93 (<s>, 2H), 1.64 (d,J= 62.5 H<z>, 6H). ESI MS [M+H]+ for C16H23CIFN5O8P2, cal. 530.1, found 530.2 Reference Example 36

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(((S)-tetrahydrofuran-3-yl)amino)-9H-purin-9-yl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0253]

[0254] The base compound was synthesized in a similar way to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d6) 68.66 (d,J= 6.2 Hz, 1H) , 8.31 (s, 1H), 6.38 (dd,J= 14.3, 4.6 Hz, 1H), 5.26 (ddd,J= 52.4, 4.2, 2.4 Hz, 1H), 4.61 -4.67 (m, 1H), 4.57 -4.45 (m, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.04 (q,J= 5.0 H<z>, 1H), 3.89 (dt,J= 15.3, 7.8 H<z>, 2H), 3.73 (q,J= 7.8 H<z>, 1H), 3.61 (dd,J=8.9, 4.4 H<z>, 1H), 2.36 - 1.99 (m, 4H). ESI MS [M-H]- for C15H20CIFN5O9P2, cal. 53o.04, found 530.1.

Reference Example 37

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(((R)-tetrahydrofuran-3-yl)amino)-9H-purin-9-yl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0255]

[0256] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: <1>H NMR (400 MH<z>, DMSO-d<6>) 68.65 ( d,J= 6.8 H<z>, 1H), 8.31 (<s>, 1H), 6.39 (dd,J= 14.2, 4.6 H<z>, 1H), 5.26 (ddd,J= 52.4, 4.3, 4.3 H<z>, 1H), 4.69 -4.56 (m, 1H), 4.51 (dt,J= 18.6, 4.6 H<z>, 1H), 4.20 (t,J= 6.1 H<z>, 2H), 4.04 (q,J= 5.0 Hz , 1H), 3.89 (dt,J= 18.6, 7.9 Hz , 2H), 3.74 (q,J= 7.8 Hz , 1H), 3.67 - 3.54 (m, 1H), 2.35 - 1.90 ( m, 4H). ESI m S [M-H]- for C15H20CIFN5O9P2, cal. 530.04, found 530.1.

Reference Example 38

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-((tetrahydro-2H-pyran-4-yl)amino)-9H-purin-9-yl)- 4-fluoro-3-h hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0257]

[0258] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MH<z>, DMSO-d a) 68.47 -8.34 (m, 1H ), 8.30 (<s>, 1H), 6.37 (dd,J= 14.1,4.8 H<z>, 1H), 5.25 (ddd,J= 52.4, 4.3, 4.3 H<z>, 1H), 4.92 - 4.65 (m, 1H), 4.59 - 4.39 (m, 1H), 4.19 (t,J= 6.2 H<z>, 2H), 4.03 (q,J= 5.1 H<z>, 1H), 3.89 (d,J = 11.3 H<z>, 2H), 3.41 (t,J= 11.4 H<z>, 2H), 2.26 (dd,J= 20.5 H<z>, 2H), 1.92 - 1.45 (m, 4H). ESI MS [M-H]- for C1aH22ClFN50gP2, cal. 544.06, found 544.1.

Reference Example 39

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(pyrrolidin-1-yl)-9H-pu/7n-9-//)-4-fluoro- 3-hydroxytetrahydrofuran-2-yl)m ethoxy)(h idroxy)phosphoryl)m ethyl)phosphonic

[0259]

[0260] The base compound was synthesized in a similar way to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 68.27 (d,J= 2.1 Hz, 1H ), 6.39 (dd,J= 14.1,4.7 Hz, 1H), 5.26 (dt,J= 52.5, 4.3 H<z>, 1H), 4.50 (dt,J= 18.5, 4.6 H<z>, 1H), 4.19 (t,J= 5.9 H<z>, 2H), 4.05 (q,J= 5.3, 4.1 H<z>, 3H), 3.60 (t,J= 6.8 Hz , 2H), 2.27 (t,J= 20.5 Hz , 2H), 2.01 (p,J= 6.7 Hz , 2H), 1.92 (q,J= 6.7 Hz , 2H).ESI MS [M+H]+for C15H21C1FN5O8P2, cal. 516.1, found 516.1

Reference Example 40

Synthesis of acid ((((2R,3R,4S,5R)-5-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran- 2-yl)m ethoxy)(hydroxy)phosphoryl)m ethyl)phosphonic

[0261]

[0262] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-da) 68.30 (d,J=2.2 Hz, 1H) , 6.39 (dd,J=14.3, 4.6 Hz , 1H), 5.26 (dt,J=52.4, 4.3 Hz , 1H), 4.50 (dt,J=18.4, 4.6 Hz , 1H), 4.19 (t,J=6.0 Hz , 2H), 4.04 (q,J=5.0 Hz , 1H), 3.88 (m, 2H), 2.27 (t,J=20.5 H<z>, 2H), 1.64 (d,J=31.0 H<z> , 8H).ESI MS [M+H]+ for C1aH23CIFN50sP2, cal. 530.1, found 530.2

Reference Example 41

Acid synthesis -hydroxytetrahydrofuran-2-yl)m ethoxy)(h idroxy)phosphoryl)m ethyl)phosphonic

[0263]

[0264] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MH<z>, DMSO-d a) 68.34 (d,J= 2.1 H<z>, 1H), 6.41 (dd,J= 13.9, 4.6 H<z>, 1H), 5.27 (dt,J=52.5, 4.3 H<z>, 1H), 4.51 (dt,J= 18.5, 4.6 H<z>, 1H), 4.19 (t,J=5.8 H<z>, 2H), 4.04 (q,J=5.1 H<z>, 1H), 3.79 - 3.67 (m, 5H), 2.26 ( t,J=20.5 Hz, 2H). ESI MS [M+H]+ for C15H21CIFN5O9P2, cal. 532.1, found 532.1

Reference Example 42

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(isoindolin-2-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran -2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0265]

[0266] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 68.37 (d, J= 2.1 Hz, 1H ), 7.48 (dt,J= 9.9, 4.7 Hz, 2H), 7.43 -7.28 (m, 2H), 6.44 (dd,J= 13.8, 4.7 Hz, 1H), 5.41 (s, 2H), 5.29 (dt, J= 52.6, 4.4 Hz, 1H), 4.98 (s, 2H), 4.54 (dt,J= 18.7, 4.7 Hz, 1H), 4.21 (t,J= 5.9 Hz, 2H), 4.05 (q,J= 4.9 Hz , 1H), 2.27 (t,J= 20.5 Hz , 2H). ESI MS [M+H]+for C19H21C1FN5O8P2, cal. 564.1, found 564.1

Reference Example 43

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-((4-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0267]

[0268] The base compound was obtained using a procedure identical to that of example 29 using the product of step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 59.O1 (t,J= 6.2 Hz , 1H), 8.32 (d,J= 2.1 Hz , 1H), 7.64 - 7.08 (m, 4H), 6.38 (dd,J= 14.3, 4.6 H<z>, 1H), 5.26 (dt,J= 52.5, 4.3 H<z>, 1H), 4.64 (q,J= 7.3, 5.4 H<z>, 2H), 4.51 (dt,J= 18.7, 4.6 Hz , 1H), 4.28 - 4.11 (m, 2H) , 4.04 (q,J= 5.1 Hz , 1H), 2.27 (t,J= 20.5 Hz , 2H). ESI MS [M-H] - for C18H20CI2FN5O8P2, cal. 584.0, found 584.1.

Reference Example 44

Synthesis of acid (((((2R,3R,4S,5R))-5-(2-chloro-6-((4-fluorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0269]

[0270] The base compound was obtained using a procedure identical to that of example 29 using the product of step a of example 29 and the corresponding amine: 1H N<m>R (400 MH<z>, DMSO-d a) 59.00 (t,J= 6.3 H<z>, 1H), 8.31 (d,J= 2.2 H<z>, 1H), 7.52 - 7.24 (m, 3H), 7.23 - 7.01 (m, 2H), 6.38 (dd ,J= 14.3, 4.6 H<z>, 1H), 5.26 (dt,J= 52.4, 4.3 H<z>, 1H), 4.72 - 4.55 (m, 2H), 4.20 (t,J= a.O Hz , 3H ), 4.04 (q,J= 5.1 Hz, 1H), 2.27 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C18H20CIF2N5O8P2, cal. 568.0, found 568.2.

Reference Example 45

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-((3-methylbenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0271]

[0272] The base compound was synthesized as a white solid (87.1 mg; 31%) similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H N<m>R (4o0 MH<z>, DMSO-d a) 58.96 (t,J= 6.3 H<z>, 1H), 8.3O (d,J= 2.2 Hz, 1H), 7.2O (t,J= 7.5 Hz, 1H) , 7.17 -7.1O (m, 2H), 7.O4 (d,J= 7.4 Hz , 1H), 6.38 (dd,J= 14.3, 4.6 H<z>, 1H), 5.25 (dt,J=52.4, 4.3 H<z>, 1H), 4.68 -4.56 (m, 2H), 4.51 (dt,J= 18.4, 4.6 H<z>, 1H), 4.19 (t,J= 6.O H<z>, 2H) , 4.O3 (q,J= 5.1 H<z>, 1H), 2.35 -2.17 (m, 2H). ESI MS [M-H]- for C19H22CIFN5O8P2, cal. 564.1, found 564.2.

Reference Example 46

Synthesis of acid (((((2R,3R,4S,5R))-5-(2-chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0273]

[0274] The base compound was synthesized as a white solid (65.1 mg; 23%) similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (4o0 MH<z> , DMSO-d e) 69.02 (t,J= 6.3 H<z>, 1H), 8.32 (d,J= 2.2 Hz , 1H), 7.42 -7.32 (m, 1H), 7.17 (t,J= 9.2 Hz , 2H), 7.07 (td,J= 8.4, 2.2 Hz , 1H), 6.39 (dd,J= 14.4, 4.6 Hz , 1H), 5.26 (dt,J= 52.5, 4.3 Hz , 1H), 4.74 - 4.60 ( m , 2H), 4.51 (dt,J= 18.5, 4.7 Hz , 1H), 4.26 - 4.13 (m, 2H), 4.04 (q,J= 5.0 H<z>, 1H), 2.27 (t,J= 20.5 H<z>, 2H). ESI MS [M-H]- for C18H19CIF2N5O8P2, cal. 568.0, found 568.2

Reference Example 47

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0275]

[0276] The base compound was synthesized as a white solid (70.6 mg; 24%) similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (4o0 MH<z> , DMSO-d e) 69.03 (t,J= 6.2 H<z>, 1H), 8.33 (d,J= 2.2 H<z>, 1H), 7.45 - 7.27 (m, 4H), 6.39 (dd,J = 14.4, 4.6 H<z>, 1H), 5.26 (dt,J= 52.4, 4.2 H<z>, 1H), 4.74 -4.58 (m, 2H), 4.51 (dt,J= 18.5, 4.6 Hz, 1H ), 4.20 (t,J= 6.1 Hz , 2H), 4.04 (q,J= 5.1 Hz , 1H), 2.27 (t,J= 20.5 Hz , 2H). ESI MS [M-H]- for C18H19CI2FN5O8P2, cal. 584.O, found 584.0

Reference Example 48

Synthesis of acid (((((2R,3R,4S,5R))-5-(2-chloro-6-((2-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0277]

[0278] The base compound was synthesized in a similar way to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-da) 68.99 (t,J= 6.1 Hz, 1H) , 8.35 (s, 1H), 7.47 (dd,J= 6.O, 3.3 Hz , 1H), 7.35 - 7.22 (m, 3H), 6.40 (dd,J= 14.2, 4.6 Hz , 1H), 5.27 (dt ,J= 52.4, 4.3 Hz , 1H), 4.73 (d,J= 5.2 Hz , 2H), 4.52 (d,J= 18.5 H<z>, 1H), 4.20 (t,J= 6.2 H<z>, 2H), 4.05 (q,J= 5.1 H<z>, 1H), 2.27 (t,J= 20.5 H<z>, 2H). ESI MS [M+H]+for C18H20CI2FN5O8P2, cal. 586.0, found 58a.1

Reference Example 49

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-((2-chlorobenzyl)(methyl)amino)-9H-pu/7n-9-//)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoriI)methiI)phosphonium

[0279]

[0280] The base compound was synthesized in a similar way to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (400 MHz, DMSO-d a) 68.32 (d, J= 37.7 Hz, 1 H), 7.55 -7.46 (m, 1 H), 7.31 (bs, 2H), 7.15 (bs, 1 H), 6.41 (d,J=14.4 H<z>, 1 H), 5.61 (bs, 1 H ), 5.26 (d,J=52.6 H<z>, 1 H), 5.00 (b, 1 H), 4.49 (bs, 1 H), 4.17 (bs, 2 H), 4.03 (bs, 1 H), 3.70 (bs, 1 H), 3.18 (bs, 2 H), 2.25 (t,J= 20.4 Hz, 2 H). ESI MS [M+H]+for C19H24CI2N5O9P2, cal. aOo.0, found 600.1.

Reference Example 50

Synthesis of acid (((((2RJ3RJ4SJ5R)-5-(2-chloro-6-((pyridin-4-ylmethyl)amino)-9H-purin-9-yl)-4-fIuoro-3-hydroxytetrahydrofuran-2- iI)methoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0281]

[0282] The base compound was synthesized similarly to example 29 using the product from step a of example 29 and the corresponding amine: 1H NMR (4OO MHz, DMSO-d a) 6 9.13 (s, 1H), 8.66 ( d,J= 5.7 Hz , 2H), 8.37 (s, 1H), 7.65 (d,J= 5.6 Hz , 2H), 6.40 (dd,J= 14.0, 4.6 Hz , 1H), 5.4O -5.O8 ( m, 1H), 4.8O (d,J= 6.1 Hz , 2H), 4.53 (d,J= 18.3 Hz , 1H), 4.19 (<s>, 2H), 4.O4 (d,J= 5.2 H< z>, 1H), 2.25 (t,J= 2O.4 H<z>, 2H). ESI MS [M+H]+for C17H2oCIFNa08P2, cal. 553.1, found 553.2

Reference Example 51

Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(phenethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0283]

[0284] The base compound was synthesized in a similar manner to example 29 using the product from step a of example 29 and the corresponding amine: <1>H NMR (400 MHz, DMSO-d a) 6 8.50 (t,J= 5.7 Hz, 1H), 8.27 (s, 1H), 7.38 - 7.13 (m, 5H), 6.37 (dd,J= 14.4, 4.7 Hz, 1H), 5.25 (dt, J= 52.4, 4.2 Hz, 1H), 4.51 (dt,J= 18.5, 4.6 Hz, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.04 (t,J= 5.1 Hz, 1H), 3.66 (d,J= 7.2 Hz, 2H), 2.92 (t,J= 7.5 Hz , 2H), 2.26 (t,J= 20.5 Hz , 2H).ESI MS [M+H]+ for C19H23CIFN5O8P2, cale. 566.1, found 5a6.1

Reference Example 52

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-meí/7-9H-pu/7n-9-//)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

[0285]

[0286] The base compound was synthesized similarly to Example 29 using 6-eIoro-2-methylpurine instead of 2,6-dieloropurine: <1>H NMR (400 MHz, DMSO-d a) 68.51 (s, 1H ), 8.23 (s, 1H), 7.44 -7.19 (m, 5H), 6.44 (dd,J= 15.0, 4.6 Hz, 1H), 5.41 -5.13 (m, 1H), 4.72 (s, 2H), 4.53 ( dd,J= 18.4, 4.7 Hz , 1H), 4.19 (t,J= 6.1 Hz , 2H), 4.04 (t,J= 5.1 Hz , 1H), 2.46 (s, 3H), 2.26 (t,J= 20.5 Hz, 2H). ESI MS [M+H]+ for C19H24FN5O8P2, cal. 532.1, found 532.2

Reference Example 53

Synthesis of (((((2R,3R,4S,5R))-5-(6-(cyclopentylamino)-2-methyl-9H-purin-9-N)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0287]

[0288] The base compound was synthesized similarly to Example 29 using 6-eIoro-2-methylIpurine instead of 2,6-dieloropurine and cielopentylamine instead of beneylamine: 1H n Mr (400 MHz, DMSO-d a) 68.56 (s, 1H), 8.30 (s, 1H), 6.45 (dd,J= 14.4, 4.6 H<z>, 1H), 5.25 (dt,J= 52.5, 4.3 H<z>, 1H), 4.53 (dt ,J= 18.3, 4.5 H<z>, 1H), 4.20 (t,J= 6.1 H<z>, 2H), 4.04 (q, J= 5.0 Hz , 1H), 2.26 (t,J= 20.5 Hz , 2H), 1.98 (s, 2H), 1.82 - 1.46 (m, aH).ESI MS [M+H]+ for C^H2aFN508P2, cal.

51o.1, found 510.2

Reference Example 54

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-(trifluoromethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2 -yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0289]

[0290] The base compound was synthesized similarly to Example 29 using 6-doro-2-trifluoromethylpurine instead of 2,6-didoropurine: 1H NMR (400 MHz, DMSO-da) 69.11 (d,J= 6.3 Hz, 1 H), 8.49 (d,J= 2.1 Hz, 1 H), 7.39 -7.35 (m, 2 H), 7.34 - 7.27 (m, 2 H), 7.25 - 7.20 (m, 1 H), 6.48 (dd ,J= 14.0, 4.7 H<z>, 1 H), 5.30 (dt,J=52.4, 4.3 H<z>, 1 H), 5.20 (bs, 1 H), 4.70 (t,J= 5.7 Hz, 1 H), 4.56 (dt,J= 18.6, 4.7 Hz , 1 H), 4.21 (t,J= 6.2 Hz , 2 H), 4.06 (q,J= 5.1 Hz , 1 H), 2.26 (t,J = 20.5 Hz, 2 H). ESI MS [M+H]+ for C19H21F4N5O8P2, cal. 586.1, found 586.2.

Reference Example 55

Synthesis of acid (((((2R,3R,4S,5R))-5-(6-(cyclopentylamino)-2-(trifluoromethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2 -yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0291]

[0292] The base compound was synthesized similarly to Example 29 using 6-eloro-2-trifluoromethylpurine instead of 2,6-dieloropurine and cielopentylamine instead of beneylamine:<1>H NMR (400 MHz, DMSO-d a ) 68.59 - 8.40 (m, 2 H), 6.47 (dd,J= 13.9, 4.7 H<z>, 1 H), 5.30 (dt,J= 52.4, 4.3 H<z>, 1 H), 5.11 (bs , 1 H), 4.52 (dd,J= 28.1, 14.1 H<z>, 2 H), 4.21 (t,J= 6.0 Hz , 2 H), 4.06 (q,J= 5.2 Hz , 1 H), 2.26 (t,J= 20.4 Hz, 2 H), 2.08 - 1.90 (m, 2 H), 1.80 - 1.50 (m, 6 H). ESI MS [M+H]+ for C17H23F4N5O8P2, cal. 564.2, found 564.1

Reference Example 56

Synthesis of (((((2R,3R,4S,5R))-5-(6-(benzylamino)-2-phenyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)m ethyl)phosphonic

[0293]

[0294] Step a: The product of step b (1) of Example 29 (750 mg, 1.25 mmol), fenylboronyl acid (229 mg, 1.88 mmol) and potassium carbonate (518 mg, 3, 75 mmol) were suspended in 3:1 THF:H20 (10.3 mL). This mixture was degassed by a N2 jet for 10 minutes. Pd(PPh3)4 (144 mg, 0.13 mmol) was then added and the resulting mixture was degassed for an additional 5 minutes, sealed and heated at 80 °C overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The raw material was composed of a mixture of mono- and di-debozoylated products that was used directly in step b.

[0295] Step b: The product from step a was dissolved in methanol (12.5 mL) and potassium carbonate (518 mg, 3.75 mmol) was added. The resulting suspension was stirred overnight at room temperature and then partitioned between EtOAc and water. The organics were washed with brine, dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, gradient from 0 to 10% MeOH and CH2Cl2) as a white solid (41 mg, 8% in two steps). ESI MS [M+H]+ for C23H22FN5O3, cal. 436.2, found 436.3.

[0296] Step c: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-d6) 68.58 (<s>, 1H), 8.44 - 8.32 (m, 2H), 8.29 (d,J= 2.4 H<z>, 1H), 7.40 - 7.50 (m, 5H), 7.31 (dd,J= 8.3, 6.9 H<z>, 2H), 7.24 -7.15 (m, 1H), 6.59 (dd,J= 15.4, 4.6 H<z>, 1H), 5.30 (dt,J= 52.4, 4.1 H<z>, 1H), 4.82 (<s>, 2H), 4.69 - 4.48 (m, 1H), 4.22 (d,J=6.6 Hz, 2H), 4.08 (q,J=5.1 Hz, 1H), 2.27 (t,J=20.5 Hz, 2H). ESI MS [M-H]- for C24H26FN5O8P2, cal. 592.1, found 592.2.

Reference Example 57

Synthesis of (((((2R,3R,4S,5R))-5-(2-benzyl-6-(benzylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)m ethyl)phosphonic

[0297]

[0298] Step a: The product of step b (1) of Example 29 (391 mg, 0.659 mmol), potassium beneyltrifluoroborate (391 mg, 1.98 mmol) and cesium carbonate (1.07 g, 3.30 mmol) were suspended in 20:1 THF:H20 (6.5 mL). This mixture was degassed by a N2 jet for 10 minutes. Pd(PPh3)2Ch (96 mg, 0.132 mmol) was then added and the resulting mixture was degassed for an additional 5 minutes, sealed and heated at 80 °C for 48 hours. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, EtOAc/Hexane) as a beige solid (174 mg, 40%).

[0299] Step b: The product from step a (174 mg, 0.265 mmol) was dissolved in methanol (2.65 mL) and potassium carbonate (110 mg, 3.75 mmol) was added. The resulting suspension was stirred at room temperature for 1.5 hours and then partitioned between EtOAc and water. The organics were washed with brine, dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, gradient from 0 to 10% MeOH and CH2Cl2) as a white solid (102 mg, 86%). ESI MS [M+H]+ for C24H24FN5O3, cal. 450.2, found 450.3.

[0300] Step<c>: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-d6) 68.54 (<s>, 1H), 8.22 (<s>, 1H), 7.61 -6.94 (m, 10H), 6.44 (dd,J= 15.1,4.6 H<z>, 1H), 5.23 (dt,J=52.4, 4.1 Hz , 1H), 4.82 - 4.40 (m, 3H), 4.18 (t,J= 6.5 Hz, 2H), 4.03 (dd,J= 10.9, 5.9 Hz, 3H), 2.26 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C25H28FN5O8P2, cal. 606.1, found 606.3.

Reference Example 58

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-(piperidin-1-ylmethyl)-9H-purin-9-yl)-4-fluoro-3 -hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0301]

[0302] Step a: The product of step a of Example 35 (10.0 g, 17.24 mmol), phenylvinyl boronic acid (3.83 g, 25.86 mmol) and sodium carbonate (5.44 mg, 51.72 mmol) were suspended in 3:1 THF:H20 (100 mL). This mixture was degassed by a N2 jet for 10 minutes. Pd(PPh3)4 (1.99 g, 1.72 mmol) was then added and the resulting mixture was degassed for a further 5 minutes, heating at reflux overnight. After cooling to room temperature, the reaction was diluted with EtOAe and washed with water and brine. The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, 5% to 5% EtOAe/Hexane) as a colorless solid (8.06 g, 72%).

[0303] Step b: A suspension of the product from step a (8.06 g, 12.04 mmol), sodium periodate (15.5 g, 72.4 mmol) and 2,6-lutidine (2.80 mL, 24.1 mmol) in 2:1 THF:H20 (127.5 mL) potassium osmate dihydrate (100 mg, 0.30 mmol) was added. The resulting slurry was stirred overnight at room temperature and then partitioned between EtOAe and water. The organics were washed briefly with water and brine, dried over MgSO4 and concentrated under reduced pressure. The base compound was obtained after column chromatography (SO 2, EtOAe/Hexane) as an off-white oil (6.74 g, 97%). ESI MS [M+H]+ for C30H28FN5O6, cal. 574.2, found 574.4.

[0304] Step<c>: 1) To a solution of the product of step b (500 mg, 0.87 mmol) in dieloroethane (4.5 mL) was added piperidine (104<u>L, 1.05 mmol ) followed by sodium triaethoxyborohydride (223 mg, 1.05 mmol) in a single serving. The reaction was stirred at room temperature overnight and then partitioned between EtOAe and water. The organics were washed with brine, dried over MgSO4 and concentrated under reduced pressure to obtain the base compound, which was used without further purification. ESI MS [M+H]+ for C35H39FN6O5, cal. 643.3, found 643.3.

[0305] Step c: 2) The above crude product was dissolved in methanol (8.7 mL) and potassium carbonate (362 mg, 2.62 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAe and water. The organics were washed with brine, dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, gradient from 0 to 100% MeOH and CH2Cl2) as a white solid (151 mg, 40% in two steps). ESI MS [M+H]+ for C21H31FN6O3, cal. 435.2, found 435.3.

[0306] Step d: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-d 6) 69.40 (<s>, 1H), 8.58 - 8.03 (m, 2H), 6.45 (dd,J= 14.1, 4.8 H<z>, 1H), 5.25 (dt,J= 52.5, 4.3 Hz, 1H), 4.59 (d,J= 16.3 Hz, 2H), 4.40 (s, 1H), 4.20 (t,J= 6.1 Hz, 2H), 4.04 (q,J= 5.1 Hz, 1H), 3.61 (s, 1H), 3.08 (<s>, 2H), 2.24 (t ,J= 20.4 H<z>, 2H), 2.06 - 1.35 (m, 10H). ESI MS [M-H]- for C22H35FN6O8P2, cal. 591.2, found 591.3.

Reference Example 59

Synthesis of acid (((((2R,3R,4S,5R))-5-(6-(ddopentylamino)-2-(methoxymethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2 -yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0307]

[0308] Step a: To a solution of the product of step b of Example 58 (1.0 g, 1.74 mmol) in dieloroethane (20 mL) was added sodium triaeethoxyborohydride (443 mg, 2.09 mmol) in a single portion. The reaction was stirred at room temperature overnight and then partitioned between EtOAe and water. The organics were washed with brine, dried over MgSO4, and concentrated under reduced pressure to obtain the title compound, which was used without further purification. ESI MS [M+H]+ for C30H30FN5O6, cal. 576.2, found 576.3.

[0309] Step b: 1) To a solution of the product from step a in dieloromethane (10 mL) at 0 ° C, T<s>Cl (436 mg, 2.29 mmol) and triethylamine (400 uL, 2 .87 mmol). The reaction was allowed to warm to room temperature and stirred overnight. The reaction was diluted with EtOAe and washed with sat. NaHC03, 10% citric acid, water and brine. The organics were dried over MgSO4 and concentrated under reduced pressure to obtain the crude base compound (1.20 g, 94% in two steps) which was used directly in the next step.

[0310] Step b: 2) To a flask loaded with crude tosylate (700 mg, 0.959 mmol) and potassium carbonate (662 mg, 4.8 mmol) methanol (10 ml) was added. The resulting suspension was stirred overnight, diluted with EtOAe, and washed with water and brine. The organics were dried over MgSO4 and concentrated under reduced pressure. The base compound (85 mg, 23%) was obtained after column chromatography (SÍO2, gradient from 0 to 15% of MeOH and CH2CI2). ESI MS [M+H]+ for C17H24FN5O4, cal. 382.2, found 382.3.

[0311] Step c: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MHz, DMSO-d 6) 68.24 (s, 1H), 6.46 (dd,J= 14.9 , 4.6 Hz , 2H), 5.24 (dt,J= 52.5, 4.2 Hz , 1H), 4.54 (dt,J= 18.3, 4.4 Hz , 2H), 4.40 (s, 2H), 4.20 (t,J= 6.1 Hz , 3H), 4.04 (t, J= 5.0 Hz , 1H), 3.37 (s, 5H), 2.26 (t,J= 20.5 Hz , 2H), 1.96 (s, 3H), 1.81 -1.41 (m, 10H) . ESI MS [M-H]- for C18H28FN5O9P2, cal. 538.1, found 538.2.

Reference Example 60

Synthesis of acid (((((2R,3R,4S,5R))-5-(6-(ddopentylamino)-2-(hydroxy(phenyl)methyl)-9H-purin-9-yl)-4-fluoro-3 -hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0312]

[0313] Step a: To a solution of the product of step b of Example 58 (330 mg, 0.58 mmol) in THF (6 mL) at -78 ° C, phenylmagnesium bromide (3.0 M / Et2O, 0.86 mL). The reaction was stirred at this temperature for 1 hour and then quenched with sat. NaHCOa. The crude reaction mixture was partitioned between EtOAc and water. The organics were washed with water and brine, dried over MgSO4 and concentrated under reduced pressure. The crude material was composed of a mono-debenzoylated isomeric product that was used directly in step b. ESI MS [M+H]+ for C29H30FN5O5, cal. 548.2, found 548.3.

[0314] Step b: The product from step a was dissolved in methanol (5.8 mL) and potassium carbonate (240 mg, 1.74 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organics were washed with brine, dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SÍO2, gradient from 0 to 10% MeOH and CH2Cl2) as a white solid (118 mg, 46% in two steps). ESI MS [M+H]+ for C22H26FN5O4, cal. 444.2, found 444.3.

[0315] Step<c>: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid (1: 1 mixture of diastereomers): 1H NMR (400 MH<z>, DMSO-d 6) 68.78 - 7.85 (m, 4H), 7.49 (<s>, 4H), 7.41 - 7.08 (m, 8H), 6.47 (dd,J= 14.8, 4.6 H<z>, 2H), 5.98 -5.39 (m, 2H ), 5.24 (dt,J= 52.4, 4.2 H<z>, 1H), 5.07 (<s>, 1H), 4.54 (d,J= 14.1 Hz , 0H), 4.39 - 3.86 (m, 6H), 2.26 (t,J= 20.5 Hz, 3H), 1.99 (d,J= 34.0 Hz, 5H), 1.65 (d,J= 52.4 Hz, 13H). ESI MS [M-H]' for C23H30FN5O9P2, cal. 600.2, found 600.3.

Reference Example 61

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-(phenylethynyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2 -yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0316]

Stage

[0317] Step a: The product of step b (1) of Example 29 (750 mg, 1.24 mmol) was suspended in DMF (8.3 mL) and Et3N (260 L) was added followed by phenylacetylene (205<u>L). This mixture was degassed by a N2 jet for 10 minutes. Subsequently, C<u>I (24 mg) and Pd(PPha)2Cl2 (44 mg) were added and the resulting mixture was heated to 80 °C overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with 10% citric acid (aqueous), water and brine. The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, EtOAc/Hexane) as a tan oil (762 mg, 92%).

[0318] Step b: The product from step a (762 mg, 1.14 mmol) was dissolved in methanol (11.4 mL) and potassium carbonate (473 mg, 3.42 mmol) was added. The resulting suspension was stirred overnight at room temperature and then partitioned between EtOAc and water. The organics were washed with brine, dried (Na2SO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SO 2, gradient from 0 to 10% MeOH and CH2CI2) as a colorless oil. ESI MS [M+H]+ for C25H22FN5O3, cal. 460.2, found 460.2.

[0319] Step<c>: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-d 6) 68.65 (<s>, 1H), 8.37 (d,J= 2.3 H<z>, 1H), 7.67 - 7.57 (m, 1H), 7.47 (td,J= 5.2, 2.1 H<z>, 2H), 7.39 - 7.29 (m, 4H), 7.28 - 7.16 (m, 1H), 6.50 (dd,J= 15.2, 4.4 H<z>, 1H), 5.28 (dt,J= 52.4, 4.1 H<z>, 1H), 4.75 (<s>, 2H ), 4.52 (d,J= 18.1 Hz , 1H), 4.20 (d,J=6.4 Hz , 2H), 4.06 (q,J= 5.0 Hz , 1H), 2.28 (t,J= 20.5 Hz , 2H). ESI MS [M-H]' for C26H26FN5O8P2, cal. 616.1, found 616.3.

Reference Example 62

Synthesis of (((((2R,3R,4S,5R))-5-(6-(benzylamino)-2-phenethyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0320]

[0321] Step a: To a solution of the product of step b of Example 61 (203 mg, 0.44 mmol) in ethanol (4.4 mL) under a nitrogen atmosphere, palladium on carbon (10% by wet weight) was added , 20 mg). The nitrogen atmosphere was displaced with hydrogen and stirred at room temperature. After stirring overnight, the reaction was diluted with EtOAc and filtered through Celite. The filtrate was concentrated under reduced pressure to obtain the title compound (161 mg, 79%), which was used without further purification. ESI MS [M+H]+ for C25H26FN5O3, cal. 464.2, found 464.4.

[0322] Step b: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-<ó>6) or 8.60 - 8.14 (m, 2H) , 7.58 - 6.91 (m, 11H), 6.44 (d,J= 15.0 H<z>, 1H), 5.22 (d,J= 52.4 H<z>, 1H), 4.71 (<s>, 2H), 4.54 (dt,J= 18.4, 4.4 H<z>, 1H), 4.19 (t,J= 6.2 H<z>, 2H), 4.11 -3.96 (m, 1H), 3.23 -2.83 (m, 5H), 2.26 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C26H30FN5O8P2, cal. 620.2, found 620.2.

Reference Example 63

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-ef/n//-9H-pur/n-9-//)-4-fluoro- 3-hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0323]

[0324] Step a: The product of step b (1) of Example 29 (2.0 g, 3.32 mmol) was suspended in DMF (7.4 mL) and diisopropylamine (2.3 mL) was added followed by of trimethylsilyl-aeethylene (703 uL, 4.98). This mixture was degassed by a N2 jet for 10 minutes. C<u>I (125 mg, 0.66 mmol) and Pd(PPh3)2Cl2 (233 mg, 0.0.33 mmol) were subsequently added and the resulting mixture was degassed for a further 5 minutes, then sealed and heated at 80 °C for 36 hours. After cooling to room temperature, the reaction was diluted with EtOAc and washed with sat. NH4CI. (aqueous), water and brine. The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The desired product was obtained after column chromatography (SÍO2, 5% to 70% EtOAe/Hexane) as a beige solid (950 mg, 43%).

[0325] Step b: The product from step a (950 mg, 1.43 mmol) was dissolved in methanol (14 mL) and potassium carbonate (592 mg, 4.29 mmol) was added. The resulting suspension was stirred overnight at room temperature and then partitioned between EtOAc and water. The organics were washed with brine, dried (Na2S04) and concentrated under reduced pressure. The desired product was obtained by column chromatography (Si02, gradient from 0 to 10% MeOH and CH2CI2) to obtain the base compounds as a white solid (230 mg, 42%). ESI MS [M+H]+ for C19H18FN5O3, cal.

384.1, found 384.2.

[0326] Step<c>: The base compound was obtained by a procedure identical to that of Example 1 to give a white solid: 1H NMR (400 MH<z>, DMSO-da) 68.65 (<s>, 1H), 8.36 (d,J= 2.2 H<z>, 1H), 7.39 - 7.26 (m, 5H), 7.28 - 7.17 (m, 1H), 6.44 (dd,J=14.8, 4.5 Hz, 1H), 5.25 (dt, J= 52.5, 4.1 Hz, 1H), 4.69 (s, 2H), 4.51 (d,J= 18.1 Hz, 1H), 4.19 (d,J= 7.1 Hz, 2H), 2.27 (t,J= 20.5 Hz, 2H). ESI MS [M-H]- for C20H22FN5O8P2, cal. 540.1, found 540.2.

Reference Example 64

Synthesis of acid [({[(2R,3S,4S,5R)-5-[6-(benzyloxy)-2-chloro-9H-purin-9-yl]-4-fluoro-3-hydroxyoxolan-2-yl ]m ethoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0327]

equiv., 60% in oil) and benzyl alcohol (10 mL). The product from step b (1) of Example 29 (1.00 g, 1.88 mmol) was added and the mixture was stirred at r.t. for 2 hours. The reaction mixture was purified directly by column chromatography (0-10% MeOH in dieloromethane) to obtain the desired product as a white solid (721 mg, 97%). ESI MS [M+H]+ for C17H17CIFN4O4, cal. 395.1, found 395.1.

[0329] Step b: The product from step a (197 mg, 0.5 mmol) was dissolved in trimethyl phosphate (2.5 mL) and cooled to 0 ° C. A solution of methyenbis(phosphonium dieIoride) (624 mg, 2.5 mmol, 5 equiv.) in trimethyl phosphate (1.5 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h and then carefully quenched at −20 °C with 0.5 M triethylammonium bicarbonate solution (3.6 mL). The mixture was stirred at -20 °C for 15 min, then stirred at 0 °C for 15 min and then stirred at r.t. for 15 min. The mixture was washed with ethyl acetate (10 mL) three times. The aqueous layer was directly purified by reverse phase HPLC (C18 column, gradient from 0 to 50% aeetonitrile and water with 0.1% TFA) to obtain the desired product as a white solid (40.2 mg, 15%). :<1>H NMR (400 MHz, DMSO-d a) 68.55 (d,J= 2.1 Hz, 1H), 7.57 - 7.51 (m, 2H), 7.46 - 7.35 (m, 3H), 6.49 (dd,J = 13.6, 4.7 Hz , 1H), 5.61 (s, 2H), 5.30 (dt,J=52.4, 4.4 Hz , 1H), 4.53 (dt,J= 18.6, 4.7 Hz , 1H), 4.21 (t,J= 6.0 Hz, 2H), 4.06 (q,J= 5.0 Hz, 1H), 2.27 (t,J= 20.6 Hz, 2H). ESI MS [M-H]- for C18H19CIFN4O9P2, cal. 551.0, found 551.2.

Reference Example 65

Synthesis of acid (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-oloro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0330]

[0331] Step a: 2,4-dieIoro-7H-pyrroIo[2,3-d]pyrimidine (350 mg, 1.86 mmol) was dissolved in 15 mL of acetonitrile and treated with CS2CO3 (788 mg, 2 .42 mmol, 1.3 equiv.). The mixture was stirred at room temperature for 60 min. 2-Deoxy-2-fluoro-a-D-arabinofuranosyl 3,5-dibenzoate bromide (787 mg, 1.86 mmol, 1 equiv.) was dissolved in 10 mL of acetonitrile and added to the mixture dropwise through an addition funnel. The mixture was allowed to stir overnight at room temperature. The mixture was filtered onto a silica gel pad and concentrated. The residue was adsorbed onto silica and purified by column chromatography (hexanes/ethyl acetate) to obtain the product as a white solid in 49% yield (480 mg).

[0332] Step b: A mixture of the product of step a (480 mg, 0.9 mmol), benzyl amine (97 mg, 0.9 mmol,), and eí3n (91 mg, 0.9 mmol,) in Anhydrous EtOH (4 mL) was stirred at 65 °C for 6 hours. Excess solvent is removed under vacuum. The residue was dried under high vacuum for 30 min. Methanol (4 mL) and K2CO3 (249 mg, 1.8 mmol) were added and stirred for 1 h. at room temperature. LCMS indicated completion of the reaction. It was filtered and the filtrate was concentrated. The residue was purified by flash column to obtain the product in quantitative yield.

[0333] Step <c>: The product from step b (360 mg, 0.91 mmol) was dissolved in trimethylphosphate (4 mL) and cooled to 0 ° C (ice bath), then added dropwise a cold solution of methylenebis(phosphonic) dichloride (801 g, 3.2 mmol, 3.5 equiv.) in trimethylphosphate (2 mL). The reaction mixture was stirred at 0 °C for 1 h, and then carefully quenched with ice-cold 0.5 M triethylammonium bicarbonate solution (11 mL) and stirred at 0 °C for 15 min, and then 1 h at room temperature. The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 40% acetonitrile and water with 0.1% TFA) to give the product as a white solid: 1H NMR (400 MH<z>, DMSO-d 6) 68.60 (t,J= 6.0 H<z>, 1H), 7.59 -7.13 (m, 6H), 6.72 (<s>, 1H), 6.49 (dd,J= 15.7, 4.5 Hz, 1H ), 5.45 -5.04 (m, 1H), 4.80 -4.57 (m, 2H), 4.42 (dt,J=18.6, 4.4 Hz, 1H), 4.19-4.15 (m, 2H), 3.98 (q,J = 5.0 H<z>, 1H), 2.26 (t,J=20.5 H<z>, 2H). ESI MS [M-H]- for C18H22CIFN4O8P2, cal. 549.1, found 549.2.

Reference Example 66

Synthesis of (((((2R,3R,4S,5R))-5-(2-doro-6-(cyclopentylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl acid )m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0334]

[0335] The base compound was synthesized similarly to Example 65 using cielopentylamine instead of beneylamine: <1>H NMR (400 MH<z>, DMSO-d<6>) 67.90 (d,J= 7.2 Hz, 1H ), 7.24 (<s>, 1H), 6.72 (d,J= 3.6 Hz, 1H), 6.47 (dd,J= 15.9, 4.4 Hz, 1H), 5.15 (dt,J= 52.6, 4.1 Hz, 1H) , 4.52 -4.35 (m , 2H), 4.15 (q,J= 6.3, 5.3 Hz , 2H), 3.97 (q,J= 5.1 Hz , 1H), 2.23 (d,J= 20.5 Hz , 1H), 1.98 ( d,J= 10.6 Hz , 2H), 1.72 (s, 2H), 1.67 - 1.45 (m , 5H). ESI MS [M-H]- for C17H24CIFN4O8P2, cal. 527.1, found 527.2.

Reference Example 67

Synthesis of ((((1-(6-(benzylamino)-9H-purin-9-yl)propan-2-yl)oxy)(hydroxy)phosphoryl)-methyl)phosphonic acid

[0336]

[0337] The base compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: <1>H NMR (400 MHz, DMSO-d<6>) 68.79 (s, 1H), 8.31 (d, J= 15.8 Hz , 2H), 7.46 -7.13 (m , 5H), 4.92 -4.62 (m , 2H), 4.49 -4.25 (m , 2H), 2.17 (td,J= 20.4, 4.8 Hz , 2H), 1.14 (d,J= 6.3 Hz, 3H). ESI MS [M+H]+ for C16H22N5O6P2, cal. 442.1, found 442.1.

Reference Example 68

Synthesis of (((2-(6-(benzylamino)-9H-purin-9-yl)propoxy)(hydroxy)phosphoryl)-methyl)phosphonic acid

[0338]

[0339] The base compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1H NMR (400 MH<z>, DMSO-d 6) 68.73 (<s>, 1H), 8.35 (<s >, 1H), 8.27 (<s>, 1H), 7.41 -7.18 (<m>, 5H), 4.96 -4.82 (<m>, 1H), 4.72 (<s>, 2H), 4.39 -4.19 (m , 2H), 2.18 (t,J= 20.5, 1.6 Hz , 2H), 1.55 (d,J=7.0 Hz , 3H). ESI MS [M+H]+ for C16H22N5O6P2, cal. 442.0, found 442.1.

Reference Example 69

Synthesis of (((((((2R,3R,4S,5R))-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2- yl)m ethoxy)(hydroxy)phosphoryl)methyl)-phosphoryl)bis(oxy)) bis(methylene) d iisoprop yl bis(carbonate)

[0340]

[0341] Methylene bisphosphonium acid (20 Mg, 0.03<m>o I, trifluoroaeetate salt of Example 66) was dissolved in 0.5<m>L of DMSO. Hunig's base (0.18<m>L, 1<m m>o I, 30 eq) was added followed by eloromethyl isopropyl carbonate (0.13<m>L, 1 m m o I, 30 eq). The reaction mixture was allowed to stir at room temperature for 5 days. The reaction mixture was purified by reverse phase HPLC (C18 column, gradient from 0 to 40% acetonitrile and water with 0.1% TFA) to give the product as a white solid in a yield of 14% (3.6 mg). 1H NMR (400 MHz, DMSO-de) 68.42 (d,J= 7.7 Hz, 1H), 8.25 (s, 1H), 6.37 (dd,J= 15.2, 4.4 Hz, 1H), 5.67 - 5.43 (m, 4H ), 5.24 (ddt,J= 52.1,7.7, 4.1 Hz , 1H), 4.79 (pd,J= 6.2, 3.8 H<z>, 2H), 4.57 -4.38 (m, 1H), 4.37 -4.19 (m, 2H), 4.06 (q,J= 5.1 H<z>, 1H), 2.68 (t,J=21.2 H<z>, 2H), 1.92 (<s>, 2H), 1.80 - 1.47 (m, 6H) , 1.28-1.15 (m, 12H).). ESI MS [M-H]- for C26H39CIFN5O14P2, cal. 760.2, found 760.3.

Reference Example 70

Synthesis of acid [({[(2S,3S,4R,5R))-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2 -yl]m ethoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0342]

[0343] The base compound was synthesized in a similar manner to Example 1. <1>H NMR (400 MHz, DMSO-d e) 6 8.42 (s, 1H), 5.88 (d,J=5.9 Hz, 1H), 4.53 -4.46 (m, 1H), 4.19 (dd,J=5.0, 3.1 Hz, 1H), 4.15 -4.06 (m, 3H), 3.17 (brs, 3H), 2.26 (t,J=20.5 Hz, 2H), 1.94 - 1.53 (m, 9H). ESI MS [M+H]+ for C17H27CIN5O9P2, cal. 542.1, found 542.2.

Reference Example 71

Synthesis of acid [({1-[(2S,3S,4R,5R)-5-[6-(benzylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl ]ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0344]

(5.6 g, 13.3 mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature for 3 h and quenched with 10% Na2S203 (20 mL) and saturated NaHC03 (50 mL). The organic layer was separated, dried over MgSO4, filtered and evaporated. The crude aldehyde was purified by column chromatography (SIO2, CH2C h^CH2Ch:MeOH, 9:1) to give a yellow solid (4.8 g, eant.). ESI MS [M+H]+ for C20H21CIN5O4, cal. 430.1, found 430.2

[0346] Step b: The product from step a (860 mg, 2.0 mmol) was dissolved in anhydrous THF (20 mL) and cooled to -780C. A solution of 3M MeMgBr in Et<2>0 (2 mL, 6 mmol, 3 equiv.) was added dropwise and the reaction mixture was stirred at -780C for 10 min, then slowly warmed to room temperature and stirred at rt for 2 h. It was quenched with saturated NH4CI (10 mL), the organic layer was separated, dried over MgS0<4>, filtered and evaporated. The crude product was used without further purification. ESI MS [M+H]+ for C21H25CIN5O4, cal. 446.2, found 446.3

[0347] Step<c>: The phosphonylation step was performed similarly to Example 1.<1>H NMR (400 MH<z>, DMSO-<ó 6>) 69.00 - 8.88 (m, 1H), 8.51 (s, 1H), 7.37 - 7.17 (m, 5H), 5.81 (d,J= 7.0 Hz, 1H), 4.70 - 4.51 (m, 4H), 4.32 - 4.25 (m, 1H), 3.83 (dd,J = 5.3, 2.6 Hz, 1H), 2.22 (t,J= 20.5 Hz, 2H), 1.26 (d,J=6.4 Hz, 3H). ESI MS [M+H]+ for C19H25CIN5O9P2, cal.

564.1, found 564.1.

Reference Example 72

Synthesis of acid [({1-[(2S,3S,4R,5R)-5-[6-(benzylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-M ]propoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0348]

[349] The base compound was synthesized similarly to Example 71. H NMR (400 MH, DMSO-d6) 68.97 - 8.91 (m, 1H), 8.53 (s, 1H), 7.38 - 7.20 (m, 5H), 5.79 (d,J=7.4 Hz, 1H), 4.65 (d,J=6.3 Hz, 2H), 4.62 - 4 .56 (m, 1H), 4.54 - 4.46 (m, 1H), 4.34 (d,J=5.5 Hz, 1H), 3.89 (dd,J=6,1,2,2 Hz , 1H), 2.22 (t,J=20.5 Hz , 2H), 1.69 (s, 1H), 1.58 (q,J=7.1 Hz , 1H), 0.90 ( t,J=7.4 Hz, 3H). ESI MS [M+H]+ for C20H27CIN5O9P2, cal. 578.1, found 578.2.

Reference Example 73

Synthesis of acid [({[(2R,3R,4R,5R))-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxy-4-methyloxolan-2 -yl]m ethoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0350]

[0351] Step a: A p-D-ribofuranose, 2-C-methiI-, 1,2,3,5-tetrabenzoate (4.0 g, 6.89 mmol, 1 equiv.) and 2,6-dieIoropurine (1 .43 g, 7.58 mmol, 1.1 equiv.) in acetonitrile (23 mL) at 0 °C, 1,8-Diazabicyclo[5.4.0]undec-7-ene (2.58 mL, 17, 23 mmol, 2.5 equiv.) followed by trimethylsilyl trifluoromethanesulfonate (5.11 mL, 28.25 mmol, 4.1 equiv.) dropwise over 5 minutes. The reaction mixture was stirred at 0 °C for 15 minutes and heated at 65 °C for 5 hours. After cooling to room temperature, the reaction was diluted with dichloromethane and washed with sat. sodium bicarbonate. here (x2) and brine (<x>1). The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The desired product was obtained after column chromatography (SÍO2, 25% to 66% EtOAc/Hexane) as a white solid (1.30 g, 97%).

[0352] Step b: 1) A product from step a (1.3 g, 2.01 mmol), cyclopentylamine (297<j>L, 3.01 mmol, 1.5 equiv.) and triethylamine (560|jL, 4.02 mmol, 2.0 equiv.). The mixture was stirred at 70 °C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure and the material obtained was used without further purification.

[0353] 2) The above product was dissolved in methanol (20 mL) and potassium carbonate (1.06 g, 7.63 mmol, 3.8 equiv.) was added. After stirring at room temperature for 2 hours, the residue was adsorbed onto celite and purified by column chromatography (<s>¡o2, 0% to 10% DCM/MeOH) as a colorless oil (612 mg, 79%, two stages).

[0354] Step c: The base compound was synthesized similarly to Example 1.1H NMR (400 MHz, DMSO-d 6) 68.38 (dd,J= 18.2, 8.1 Hz, 1 H), 8.26 (d,J= 10.0 Hz , 1 H), 5.86 (s, 1 H), 4.42 (q,J= 7.2 Hz , 1 H), 4.27 (h,J= 10.6, 10.0 Hz , 2 H), 4.06 (s, 3 H), 2.28 (t,J= 20.4 Hz, 2 H), 1.93 (d,J=16.3 Hz, 2 H), 1.78 - 1.43 (m, 6 H). ESI MS [M-H]- for C17H25CIN5O9P2, cal. 540.1, found 540.2.

Reference Example 74

Synthesis of methyl ((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)hydrogen phosphonate ((h hydroxy(methoxy)phosphoryl)methyl)

[0355]

[0356] Step a: The nucleoside (2.0 g, 5.4 mmol) was dissolved in trimethylphosphate (30 mL) and cooled to 0 ° C (ice bath), then a cold solution of methylenbis(phosphonium dieIoride) (4.0 g, 16.2 mmol, 3 equiv.) in trimethylphosphate (15 mL). The reaction mixture was stirred at 0°C for 2h, then cooled to approximately -40°C and anhydrous MeOH (30 mL) was added and slowly warmed to room temperature. The reaction mixture was neutralized with saturated NaHCOa (80 mL) and diluted with water (150 mL) and EtOAe (150 mL). The organic layer was separated, dried over MgSO4, filtered and evaporated. The product was purified first by column chromatography (SÍO2, EtOAe^EtOAe:MeOH, 8:2) and then by HPLC R<p>18 (H2O+0.1o/o TFA/aeetonitriIo+0.1% TFA) to give the desired product as a white solid in 11% yield (405 mg). ESI MS [M+H]+ for C19H31CIN5O9P2, cal. 570.1, found 570.2.

[0357] Step b: To a solution of the product from step a (75 mg, 0.13 mmol) in acetone (1 mL), sodium iodide (50 mg, 0.33 mmol) was added. This solution was heated at 65 °C for 6 h. The solvent was evaporated; The residue was dissolved in water and purified by reverse phase HPLC (C18 column, gradient from 0 to 40% acetonitrile and water with 0.1% TFA) to give the product as a white solid in 62% yield ( 51 mg). 1H NMR (400 MH<z>, DMSO-d 6) 68.41 (d,J= 2.0 H<z>, 1H), 8.36 (d,J= 7.8 H<z>, 1H), 5.85 (d,J= 5.6 H<z>, 1H), 5.04 (brs, 1H), 4.53 (t,J= 5.5 H<z>, 1H), 4.47 -4.34 (m, 1H), 4.24 - 3.95 (m, 4H), 3.58 (d,J= 11.3 H<z>, 2H), 2.37 (dd,J= 20.5, 20.5 H<z>, 2H), 2.07 - 1.36 (m, 8H). ESI MS [M+H]+ for C17H26CIN5O9P2, cal. 542.8, found 542.2.

Reference Example 75

Synthesis of [(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methyl phenyl [(diphenoxyphosphoryl)m ethyl]phosphonate

[0358]

A cold solution of methyenbis(phosphonic dieIoride) (375 mg, 1.5 mmol, 1.5 equiv.) in trimethylphosphate (3 mL) was added dropwise and the reaction mixture was stirred at 0 °C for 3 h. Solid phenol (470 mg, 5 mmol, 5 equiv.) was added and once dissolved, TEA (835 pL, 6 mmol, 6 equiv.) was added dropwise. The mixture was stirred at 0°C for 15 minutes and then at room temperature overnight. It was diluted with H2O (15 mL) and the product was extracted with MTBE (2 * 10 mL). The combined organics were dried over MgS0<4>, filtered and evaporated. The crude product was purified by column chromatography (SÍO2, H e x^ 100% EtOAc) to give a white solid (80 mg, 10%).<1>H NMR (4o0 MHz, DMSO-d<6>) 68.38 ( d,J= 4.3 Hz, 1H), 7.41 -7.33 (m, 4H), 7.32 -7.25 (m, 2H), 7.25 -7.11 (m, 9H), 5.89 (dd,J= 5.3, 3.2 Hz, 1H) , 5.63 (dd,J= 6.0, 4.4 Hz, 1H), 5.47 - 5.41 (m, 1H), 4.62 - 4.54 (m, 1H), 4.49 - 4.32 (m, 2H), 4.28 - 4.08 (m, 1H) , 3.67 - 3.47 (m, 2H), 3.35 (s, 3H), 1.90 -1.52 (m, 8H). ESI MS [M+H]+ for C35H39CIN5O9P2, cal. 770.2, found 770.3

Reference Example 76

Synthesis deb is(3-chlorophenyl) [({[(2RJ3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)-amino]-9H-purin-9-yl}-3,4 -dih idroxyoxolan-2-yl]methoxy}(3-chlorophenoxy)phosphoryl)-m ethyl]phosphonate

[0360]

[0361] The base compound was synthesized similarly to Example 75.<1>H NMR (400 MHz, DMSO-d<6>) 6 8.32 (s, 1H), 7.43 -7.34 (m, 2H), 7.34 - 7.05 (m, 10H), 5.88 (t,J=4.7 Hz, 1H), 5.62 (s, 1H), 5.43 (s, 1H), 4.59 -4.36 (m, 3H), 4.26 -4.10 (m, 2H) , 3.86 - 3.70 (m, 2H), 3.04 (s, 3H), 1.90 - 1.46 (m, 8H). ESI MS [M+H]+ for C35H36CI4N5O9P2, cal. 872.1, found 872.2.

Reference Example 77

Synthesis debis(3,4-dichlorophenyl) [({[(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3 ,4-d ihydroxyoxolan-2-yl]methoxy}(3,4-dichlorophenoxy)phosphoryl)m ethyl]phosphonate

[0362]

[0363] The base compound was synthesized similarly to Example 75.<1>H NMR (400 MHz, DMSO-d e) 6 8.27 (s, 1H), 7.65 - 7.57 (m, 2H), 7.52 - 7.40 ( <m>, 3H), 7.32 - 7.04 (<m>, 4H), 5.87 (t,J= 5.0 H<z>, 1H), 5.63 (t,J= 6.1 Hz, 1H), 5.43 (dd,J = 5.6, 3.7 Hz, 1H), 4.59 - 4.35 (m, 3H), 4.30 - 4.08 (<m>, 2H), 3.85 (t,J= 22.0 H<z>, 2H), 3.32 (<s>, 3H), 1.89 - 1.45 (m, 8H). ESI MS [M+H]+ for C35H33CI7N5O9P2, cal. 974.0, found 974.2.

Reference Example 78

Synthesis of 2-({[(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2 Methyl -M]methoxy}[2-(methoxycarbonyl)phenoxy]phosphorM)-metM][2-(methoxycarbonyl)phenoxy}oxy)benzoate

[0364]

[0365] The base compound was synthesized similarly to Example 75. H NMR (400 MH, DMSO-de) 68.31 - 8.27 (m, 1H), 7.82 -7.74 (m , 3H), 7.57 -7.49 (m, 2H), 7.46 -7.39 (m, 1H), 7.36 -7.17 (m, 6H), 5.83 (t,J = 5.9 Hz, 1H), 5.60 -5.52 (m, 1H), 5.37 (s, 1H), 4.55 - 4.29 (m, 3H), 4.15 - 4.04 ( m, 2H), 3.81 - 3.74 (m, 2H), 3.72 - 3.65 (m, 3H), 3.32 (s, 9H), 1.88 - 1.47 (m, 8H). ESI MS [M+H]+ for C41H45CIN5O15P2, cal. 944.2, found 944.3.

Reference Example 79

Synthesis of acid [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1S)-1-(4-fluorophenN)etN]amino}-9H-purin-9- N)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]-phosphonic

[0366]

[0367] The base compound was synthesized in a similar manner to Example 29.<1>H NMR (400 MHz, DMSO-d e) 68.92 (d,J= 8.3 Hz, 1H), 8.28 (s, 1H), 7.45 ( bs, 2H), 7.11 (td,J= 9.1, 1.4 Hz, 2H), 6.34 (dd,J= 14.3, 4.6 Hz, 1H), 5.39 (bs, 1H), 5.31 -5.12 (m, 1H), 5.14 (bs, 1H), 4.48 (dt,J= 18.5, 4.5 Hz, 1H), 4.17 (s, 3H), 4.01 (d,J= 5.2 Hz, 2H), 2.24 (t,J= 20.4 Hz, 3H) , 1.51 (d,J= 7.0 H<z>, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2

Reference Example 80

Synthesis of acid [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1R)-1-phenylethyl]amino}-9H-purin-9-yl)-4- fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0368]

[0369] The base compound was synthesized similarly to Example 29. <1>H NMR (400 MH<z>, DMSO-d<6>) 68.90 (d,J= 8.3 H<z>, 1H), 8.28 (<s>, 1H), 7.42 (d,J= 7.6 H<z>, 2H), 7.29 (t,J= 7.5 H<z>, 2H), 7.19 (bs, 1H), 6.34 (dd,J = 14.8, 4.4 H<z>, 1H), 5.39 (bs, 1H), 5.23 (d,J= 52.6 H<z>, 1H), 4.49 (d,J= 19.2 H<z>, 2H), 4.17 (bs, 2H), 4.01 (d,J= 5.1 H<z>, 1H), 2.24 (t,J= 20.6 H<z>, 2H), 1.52 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O8P2, cal. 566.1, found 566.1

Reference Example 81

Synthesis of acid [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1S)-1-phenylethyl]amino}-9H-purin-9-yl)-4- fluoro-3-hydroxyoxolan-2-yl]m ethoxyl(hydroxy)phosphoryl)m ethyl]phosphonic

[0370]

[0371] The base compound was synthesized in a similar manner to Example 29. <1>H NMR (400 MH<z>, DMSO-d<6>) 68.91 (d,J= 8.3 Hz, 1H), 8.28 (s, 1H), 7.41 (d,J= 7.5 Hz , 2H), 7.29 (t,J= 7.6 Hz , 2H), 7.20 (d,J= 7.6 Hz , 1H), 6.34 (d,J= 14.1 Hz , 1H) , 5.39 (bs, 1H), 5.21 (d,J= 52.5 H<z>, 1H), 4.47 (d,J= 18.3 H<z>, 2H), 4.17 (<s>, 2H), 4.01 (< s>, 1H), 2.24 (t,J= 20.6 H<z>, 2H), 1.52 (d,J= 7.1 Hz , 3H). ESI MS [M+H]+ for C19H24CIFN5O8P2, cal. 566.1, found 566.1

Reference Example 82

Synthesis of acid [({[(2R,3R,4S,SR)-5-[6-(cyclopentylamino)-2-[hydroxy(oxan-4-yl)methyl]-9H-purin-9-yl]-4 -fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]-phosphonic

[0372]

[0373] Step a: The product from step b of Example 58 (1.00 g, 1.75 mmol) was dissolved in THF (9 mL) and cooled to -78 0C. 4-Tetrahydropyranylmagnesium bromide (9 mL, 8.75 mmol, 0.2M in THF) was added dropwise. The reaction mixture was allowed to warm to r.t. and stirred to t.r. for 3h. The reaction mixture was cooled to 0 °C, methanol (50 mL) was added and stirred at r.t. for 14h. The reaction mixture was loaded dry onto silica gel and purified by silica gel chromatography (0-10% MeOH in DCM) to obtain the desired product as a white solid (273 mg, 35%).

[0374] Step b: The base compound was synthesized as a white solid (44 mg; 29%) in a similar manner to Example 29. <1>H NMR (400 MHz, DMSO-da) 66.60 -6.40 (m, 1H) , 5.26 (d,J=53.3 Hz, 1H), 4.63 -4.39 (m, 2H), 4.30 -4.13 (m, 2H), 4.13 -3.97 (m, 1H), 3.94 -3.75 (m, 2H), 3.38 -3.13 (m, 2H), 2.26 (t,J= 20.4 Hz, 2H), 2.17 -1.85 (m, 3H), 1.85 - 1.22 (m, 12H). ESI m S [M-h ]- for C23H35FN5O9P2, cal. 606.2, found 6o6.3.

Reference Example 83 Synthesis of acid (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino}-9H-purin-9-yl)-4-fluoro -3-hydroxytetrahydrofuran-2-yl)m ethoxy)(hydroxy)phosphoryl)methyl)phosphonic

[0375]

[0376] This compound was obtained in a similar manner to Example 29.<1>H NMR (400 MHz, DMSO-d a) 68.7 (brs, 2H), 8.30 (d,J= 2.1 Hz, 1H), 6.4o ( dd,J= 14.3, 4.6 Hz, 1H), 6.09 (brs, 1H), 5.25 (dt,J= 52.5, 4.3 Hz, 1H), 4.53-4.43 (m, 1H), 4.23 4.14 (m, 2H), 4.09 - 3.98 (m, 1H), 2.28 (dd,J= 20.5 Hz, J= 20.5 Hz, 2H), 2.5 (s, 3H), 1.96 - 1.44 (m, 9H). ESI MS [M+h ]+ for C17H25CIFN5O8P2, cal. 544.8, found: 544.2

Reference Example 84

Synthesis of ((2R,3R,4S,SR)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl )methyl hydrogen ((hydroxy(methoxy)phosphoryl)methyl)phosphonate

[0377]

[0378] Step a: 2-Chloropurine fluoriboside (579 mg, 1.5 mmol) was dissolved in trimethyl phosphate (7.5 mL) and cooled to 0 ° C (ice bath), then added drop drop a cold solution of methylenebis(phosphonic) dichloride (1.87 g, 7.5 mmol, 5 equiv.) in trimethyl phosphate (4.5 mL). The reaction mixture was stirred at 0 °C for 3 h, and then carefully quenched with methanol (7 mL) and stirred at 0 °C for 30 min, then 1 h at room temperature, and then 3 h at 40 °C. The reaction mixture was concentrated in vacuo and dissolved in ethyl acetate (20 mL). The organic layer was washed with saturated aqueous NaHCOa, dried over sodium sulfate and evaporated to dryness. The residue was purified by column chromatography (methanol in dichloromethane gradient from 0 to 10%) to obtain the desired product as a pale yellow solid (701 mg, 80%).<1>H NMR (400 MHz, DMSO-d a ) 68.32 (dd,J=12.0, 2.3 Hz , 1H), 6.42 (dd,J=15.5, 4.4 Hz , 1H), 6.15 (t,J=4.8 Hz , 1H), 5.43 - 5.07 (m, 1H), 4.60 - 4.39 (m, 1H), 4.27 (q,J= 7.3, 5.7 Hz, 2H), 4.12 - 4.03 (dq,J= 9.6, 5.3 Hz, 1H), 3.69 - 3.59 (m, 9H), 2.96 - 2.74 (m, 2H), 2.50 (s, 3H), 2.04 - 1.42 (m,9H). ESI MS [M+H]+ for C20H31CIFN5O8P2, cal. 586.9, found 586.2.

[0379] Step b: To a solution of the product from step a (58 mg, 0.1 mmol) in acetone (1 mL), sodium iodide (75 mg, 0.5 mmol) was added. This solution was heated at 60 °C for 24 h. The solvent was evaporated, the residue was dissolved in water and purified by reverse phase HPLC (C18 column, gradient from 0 to 30% aeetonitrile and water with 0.1% TFA) to give the product as a white solid in a 65% yield (42 mg).<1>H NMR (400 MHz, DMSO-d<6>) 68.31 (d,J= 2.2 Hz, 1H), 6.40 (dd,J= 14.6, 4.6 Hz, 1H) , 5.25 (dt,J= 52.4, 4.2 Hz , 1H), 4.48 (dt,J= 18.3, 4.4 Hz , 1H), 4.18 (t,J= 6.1 Hz , 2H), 4.04 (m, 2H), 3.58 ( d,J= 11.2 Hz, 3H), 2.5 (s, 3H). 2.39 (dd,J= 20.4 Hz ,J= 20.4 Hz , 2H), 2.00 - 1.42 (m, 9H). ESI MS [M+H]+ for C18H27C1FN5O8P2, cal. 558.8, found 558.2.

Reference Example 85

Synthesis of ((2R,3R,4S,SR)-5-(2-chloro-6-(cidopentyl(methyl)amino)-9H-purin-9-M}-4-fluoro-3-hydroxytetrahydrofuran-2-yl )methyl hydrogen ((dimethoxyphosphoryl)methyl) phosphonate and Synthesis of (((((2R,3R,4S,5R)-5-(2-doro-6-(ddopentyl(methyl)am ino)-9H-purin- Methyl hydrogen 9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(methoxy)phosphoryl)methyl)phosphonate

[0380]

[0381] Step a: To a solution of the product of example 83, step a (150 mg, 0.26 mmol) in acetone (3 mL), sodium iodide (40 mg, 0.26 mmol) was added. This solution was stirred at room temperature for 24 h. The solvent was evaporated, the residue was dissolved in water and purified by reverse phase HPLC (C18 column, gradient from 0 to 30% acetonitrile and water with 0.1% TFA) to give ((2R,3R,4S,5R )-5-(2-cIoro-6-(cycIopentiIo(methiI)amino)-9H-purin-9-iI)-4-fIuoro-3-hydroxytetrahydrofuran-2-yl)methyl hydrogen ((dimethoxyphosphoryl)methyl)- phosphonate as white solid in 20% yield (35 mg). 1H NMR (400 MHz, DMSO-d6) 68.31 (d,J= 2.2 Hz, 1H), 6.41 (dd,J=14.9, 4.5 Hz, 1H), 5.25 (dt,J=52.3, 4.1 Hz, 1H), 4.53-4.43 (m, 1H), 4.24 - 4.12 (m, 2H), 4.08-4.02 (m, 1H), 3.66 (d,J=2.0 Hz, 3H), 3.63 (d,J=2.0 Hz, 3H) , 2.60 (dd,J=20.8 Hz ,J=20.8 Hz , 2H), 2.50 (s, 3H), 2.01 - 1.55 (m, 9H). ESI MS [M+H]+for C19H29CIFN5O8P2, cal. 572.9, found 572.3.

[0382] (((((2R,3R,4S,5R)-5-(2-cIoro-6-(cycIopentiIo(methiI)amino)-9H-purin-9-iI)-4-fIuoro-3-hydroxytetrahydrofuran -2-iI)methoxy)(methoxy)phosphoriI)methiI)-methyl hydrogen phosphonate as a white solid as a 1:1 mixture of diastereoisomers in 30% yield (52 mg). 1H NMR (400 MHz, DMSO-d 6) 68.34 (d,J= 2.3 Hz, 0.5H, 1st day), 8.30 (d,J= 2.3 Hz, 0.5H, 2nd day), 6.54 -6.32 (m, 1H ), 5.38 -5.11 (m, 1H), 4.59 -4.39 (m, 1H), 4.26 (m, 2H), 4.07 (m, 1H), 3.64 (d,J= 11.3 H<z>, 3H), 3.59 (d,J= 11.2, 1.5H, 1st day), 3.59 (d,J= 11.2, 1.5H, 1st day), 2.69-2.53 (m, 2H), 2.5 (<s>, 3H), 1.97 - 1.52 (m, 9H). ESI MS [M+H]+ for C19H29CIFN5O8P2, cal. 572.9, found 572.2.

Reference Example 86

Synthesis of acid (((((2R,3R,4S,5R))-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl}-4-fluoro-3-hydroxytetrahydrofuran -2-yl)methoxy)(methoxy)phosphoryl)m ethyl)phosphonic

[0383]

[0384] This compound was obtained as a 1:1 mixture of diastereoisomers in a similar manner to Example 84. 1H NMR (400 MHz, DMSO-d6) 68.34 (d,J= 2.3 Hz, 0.5H, 1o day), 8.29 ( d,J= 2.3 Hz, 0.5H, 2nd day), 6.52 -6.32 (m, 1H), 6.07 (brs, 1H), 5.34 -5.14 (m, 1H), 4.56 -4.43 (m, 1H), 4.30 - 4.21 (m, 2H), 4.11 -4.03 (m, 1H), 3.63 (d,J= 11.2 H<z>, 1.5H, 10day), 3.63 (d,J= 11.2 Hz , 1.5H, 2ndday), 2.50 (s, 3H), 2.48 - 2.34 (m, 2H), 1.92 - 1.53 (m, 9H). ESI MS [M+H]+for C18H27CIFN5O8P2, cal. 558.3, found 558.2.

Example 87

Synthesis of [({[(2R,3S,4R,5R))-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4 acid -dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0385]

[0386] Step a: 4,6-didoro-1H-pyrazolo[3,4-d]pyrimidine (25 g, 132 mmol) and ammonium sulfate (0.20 g, 1.5 mmol) were dissolved in 150 mL of hexamethyldisilziane. The mixture was heated to reflux and stirred for 3 hours. The mixture was then concentrated to dryness. The solid residue was taken up in 300 mL of acetonitrile and the protected ribose (50.6 g, 159 mmol) was added. This mixture was cooled to 0°C and TMSOTf (27 mL, 145 mmol) was added dropwise. The mixture was then warmed to room temperature and allowed to stir overnight. The mixture was then concentrated and taken up in ethyl acetate. The organics were washed with saturated NaHC0<3>and brine. The organics were dried with MgS0<4>, filtered and concentrated. The crude residue was purified by column chromatography (hexanes/ethyl acetate) to obtain the desired compound (48 g, 108 mmol) in 82% overall yield. H NMR (400 MH, DMSO- d<6>) 68.75 (s, 1H), 6.47 (d,J= 3.2 Hz, 1H), 5.82 (dd,J= 5.3, 3.2 Hz, 1H), 5.63 (t,J= 5.8 Hz, 1H), 4.47 - 4.40 (m, 1H), 4.37 - 4.30 (m, 1H), 4.12 - 4.02 (m, 1H), 2.09 (s, 3H), 2.06 (s, 3H), 1.97 (s, 3H). ESI MS [M+Na]+ for C16H16CI2N4N<a>O7, cal. 469.0, found 469.0.

[0387] Step b: The product from step a (22 g, 49.3 mmol) was dissolved in MeOH (100 mL) and cooled to 0 °C. Cielopentylamine (5.1 g, 51.8 mmol, 1.05 equiv.) and triethylamine (7.2 mL, 51.8 mmol, 1.05 equiv.) were added and the reaction mixture was stirred at 0 °C. for 15 min and then at rt for 4 h. 7M NH3 in MeOH (60 mL) was added and the reaction was stirred at rt for 1 day. The reaction mixture was evaporated and the crude product was used in the next step without purification. ESI MS [M+H]+ for C15H21CIN5O4, cal. 370.1, found 370.2.

[0388] Step c: The phosphonylation step was carried out similarly to Example 1. <1>H NMR (400 MH<z>, DMSO-d<6>) 6 8.68 (d,J= 7.2 Hz, 1H), 8.24 (s, 1H), 6.00 (d,J= 4.2 Hz, 1H), 4.49 (t,J= 4.7 Hz, 1H), 4.41 (q,J= 6.7 Hz, 1H), 4.26 (t, J= 4.7 Hz, 1H), 4.15 -4.00 (<m>, 2H), 3.94 - 3.84 (m, 1H), 2.16 (t,J= 20.5 H<z>, 2H), 2.04 - 1.91 (m, 2H ), 1.79 - 1.45 (m, 6H). ESI m S [M+H]+ for C16H25CIN5O9P2, cal. 528.1, found 528.2.

Example 88

Synthesis of [({[(2R,3S,4R,5R))-5-[4-(benzylamino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4 acid -dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0389]

[0390] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-de) 69.38 - 9.18 (m, 1H), 8.35 -8.16 (m, 1H), 7.39 -7.19 ( m, 5H), 6.07 -5.94 (m, 1H), 4.69 (d,J= 5.4 Hz, 2H), 4.58 -4.44 (m, 1H), 4.30 -4.20 (m, 1H), 4.15 - 4.01 (m, 2H), 3.96 - 3.80 (m, 1H), 2.17 (t,J= 20.9 Hz, 2H). ESI MS [M-H]- for C18H22CIN5O9P2, cal.

548.1, found 548.1.

Example 89

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S}-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidine acid -1-yl}-3,4-dih idroxyoxolan-2-yl]m ethoxyl(hydroxy)phosphoryl)m ethyl] phosphonic

[0391]

[0392] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-de) 69.26 - 8.95 (m, 1H), 8.35 -8.17 (m, 1H), 7.48 -7.28 ( m, 4H), 7.28 -7.09 (<m>, 1H), 6.09 -5.87 (m, 1H), 5.42 (q,J= 6.9 Hz, 1H), 4.60 -4.33 (m, 1H), 4.33 -4.16 ( m, 1H), 4.13 -3.96 (m, 2H), 3.97 - 3.80 (<m>, 1H), 2.35 - 1.95 (m, 2H), 1.62 - 1.36 (m, 3H). ESI MS [M-H]- for C19H24CIN5O9P2, cal. 562.1, found 562.2.

Example 90

Synthesis of [({[(2R,3S,4R,5R))-5-(6-doro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidine acid -1-yl}-3,4-dih idroxyoxolan-2-yl]m ethoxyl(h idroxy}phosphoryl}m ethyl]phosphonic

[0393]

[0394] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-d e) 69.16 (d,J= 8.4 Hz, 1H), 8.32 (s, 1H), 7.48 - 7.30 (m, 4H), 7.28 -7.15 (m, 1H), 6.09 -5.79 (m, 1H), 5.47 -5.36 (m, 1H), 4.58 -4.42 (m, 1H), 4.32 - 4.19 (m, 1H) ), 4.17 - 3.95 (m, 2H), 3.95 - 3.79 (m, 1H), 2.18 (t,J= 20.8 Hz, 2H), 1.71 - 1.37 (m, 4H). ESI MS [M-H]- for C19H24CIN5O9P2, cal. 562.1, found 562.2.

Example 91

Synthesis of acid [({[(2R,3S,4R,5R}-5-(6-chloro-4-{[(4-dorophenyl}metN]amino}-1H-pyrazolo[3,4-d]pyrimidin- 1-N}-3,4-dih idroxyoxolan-2-yl]m ethoxy}(h idroxy}phosphoryl}m ethyl]phosphonic

[0395]

[0396] The base compound was synthesized similarly to Example 87. H NMR (400 MHz, DMSO-de) 69.41 - 9.19 (m, 1H), 8.32 - 8.17 (m, 1H), 7.43 - 7.30 ( m, 4H), 6.02 (d,J=2.9 Hz, 1H), 4.68 (d,J=4.4 Hz, 2H), 4.56 - 4.45 (m, 1H), 4.33 -4.18 (m, 1H), 4.13 -3.80 (m, 2H), 3.62 -3.44 (m, 1H), 2.17 (t,J=20.4 Hz, 1H). ESI MS [M-H]- for C18H21CI2N5O9P2, cal.

582.0, found 582.0.

Example 92

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S}-1-(2-fluorophenyl}ethyl]amino}-1H-pyrazolo acid[3, 4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0397]

[0398] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-d e) 69.28 - 9.15 (m, 1H), 8.33 (dd,J=1.5, 0.7 Hz, 1H ), 7.43 (t,J= 7.8 Hz, 1H), 7.29 (dd,J=7.8, 5.6 Hz, 1H), 7.23 - 7.08 (m, 2H), 6.00 (d,J=4.2 Hz, 1H), 5.65 - 5.51 (m, 1H), 4.48 (t,J= 4.9 Hz, 1H), 4.26 (t,J= 4.5 Hz, 1H), 4.05 (dq,J= 10.1, 5.9, 5.2 Hz, 2H), 3.88 ( dt,J= 11.3, 6.0 Hz, 1H), 2.29 - 2.08 (t,J=20.4 Hz, 2H), 1.53 (d,J=6.8 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1

Example 93

Synthesis of [({[(2R,3S,4R,5R))-5-(6-doro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0399]

[0400] The base compound was synthesized in a similar manner to Example 87. <1>H NMR (400 MHz, DMSO-d e) 69.23 (d,J= 7.6 Hz, 1H), 8.34 (<s>, 1H), 7.44 (t,J= 7.8 Hz, 1H), 7.30 (q,J= 7.0 Hz, 1H), 7.18 (dt,J= 9.4, 6.4 Hz, 2H), 6.00 (d,J= 4.3 Hz, 1H), 5.60 (q,J= 7.1 Hz, 1H), 4.51 (t,J= 4.6 Hz, 1H), 4.26 (t,J= 4.6 Hz, 1H), 4.05 (tt,J= 10.1, 5.8 Hz, 2H), 3.88 (dd,J= 11.0, 6.2 Hz, 1H), 2.17 (t,J=20.4 Hz, 2H), 1.53 (d,J=6.7 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1

Example 94

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S}-1-(3-fluorophenyl}ethyl]amino}-1H-pyrazolo acid[3, 4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxyl(hydroxy)-phosphoryl)methyl]phosphonic

[0401]

[0402] The base compound was synthesized in a similar manner to Example 87.<1>H NMR (400 MHz, DMSO-d e) 69.17 (d,J= 7.9 Hz, 1H), 8.31 (s, 1H), 7.50 - 7.30 (m , 1H), 7.22 (d,J= 8.2 Hz, 2H), 7.06 (td,J= 8.7, 2.5 Hz, 1H), 6.00 (d,J= 4.2 Hz, 1H), 5.41 (t,J = 7.3 Hz, 1H), 4.48 (t,J= 4.7 Hz, 1H), 4.26 (t,J= 4.8 Hz, 1H), 4.05 (dq,J= 11.7, 6.5 Hz, 2H), 3.88 (dt,J = 11.2, 6.2 Hz, 1H), 2.17 (t,J= 20.5 Hz, 2H), 1.53 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1

Example 95

Synthesis of acid [({[(2R,3S,4R,5R}-5-(6-doro-4-{[(1R)-1-(3-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-N)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0403]

[0404] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-de) 69.18 (d,J= 7.9 Hz, 1H), 8.31 (t,J= 0.9 Hz, 1H ), 7.43 -7.32 (m, 1H), 7.23 (d,J= 8.8 Hz, 2H), 7.07 (t,J= 8.6 Hz, 1H), 6.00 (d,J= 4.3 Hz, 1H), 5.42 (t ,J= 7.3 Hz, 1H), 4.51 (t,J= 4.5 Hz, 1H), 4.26 (t,J= 4.7 Hz, 1H), 4.11 -3.98 (m, 2H), 3.88 (t,J= 8.6 Hz , 1H), 2.17 (t,J= 20.5 Hz , 2H), 1.52 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1 Example 96

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0405]

[0406] The base compound was synthesized in a similar manner to Example 87. <1>H NMR (400 MHz, DMSO-d e) 69.16 (d,J= 7.9 Hz, 1H), 8.30 (d,J= 1.2 Hz, 1H), 7.42 (dd,J= 8.4, 5.4 Hz, 2H), 7.15 (td,J= 8.9, 1.2 Hz, 2H), 6.00 (d,J= 4.2 Hz, 1H), 5.40 (t,J= 7.3 Hz, 1H), 4.48 (t,J= 4.8 Hz, 1H), 4.25 (t,J= 4.5 Hz, 1H), 4.18 - 3.95 (m, 2H), 3.95 - 3.82 (m, 1H), 2.16 (t ,J= 20.4 Hz , 2H), 1.52 (d,J= 7.2 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1 Example 97

Synthesis of [({[(2R,3S,4R,5R))-5-(6-doro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)-fbsfbril)m ethyl]fbsphonic

[0407]

[0408] The base compound was synthesized in a similar manner to Example 87. <1>H NMR (400 MHz, DMSO-d e) 69.16 (d,J= 7.9 Hz, 1H), 8.30 (t,J= 0.9 Hz, 1H), 7.42 (dt,J= 6.1,3.2 Hz, 2H), 7.23 -7.08 (m , 2H), 6.00 (d,J= 4.3 Hz, 1H), 5.40 (t,J= 7.2 Hz, 1H), 4.50 (t,J= 4.5 Hz, 1H), 4.26 (t,J= 4.7 Hz, 1H), 4.15 -3.98 (m, 2H), 3.87 (q,J= 8.1,5.5 Hz, 1H), 2.16 (t ,J= 20.4 Hz , 2H), 1.52 (d,J= 6.9 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O9P2, cal. 582.1, found 582.1

Example 98

Synthesis of acid ({[(2R,3S,4R,5R)-5-(6-doro-4-{[(2-dorophenyl)methyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1 -yl)-3,4-dih idroxyoxolan-2-yl]m ethoxyl(h idroxy)-phosphoryl)m ethyl]phosphonic

[0409]

[0410] The base compound was synthesized in a similar manner to Example 87.<1>H NMR (400 MHz, DMSO-d e) 6 9.29 (s, 1H), 8.29 (d,J= 1.9 Hz, 1H), 7.48 (dd,J= 5.9, 3.1 Hz, 1H), 7.43 (d,J= 5.9 Hz, 1H), 7.32 (dt,J= 6.6, 2.5 Hz, 2H), 6.07 -6.00 (m, 1H), 4.84 - 4.69 (m, 2H), 4.51 (d,J= 5.0 H<z>, 1H), 4.27 (<s>, 1H), 4.06 (<s>, 2H), 3.89 (<s>, 1H), 2.16 (t,J= 20.6 H<z>, 2H). ESI m S [M+H]+ for C18H22CI2N5O9P2, cal. 584.0, found 584.1

Example 99

Synthesis of acid [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(2-chlorophenyl)methyl](methyl)amino}-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0411]

[0412] The base compound was synthesized in a similar manner to Example 87.<1>H NMR (400 MHz, DMSO-de) 6 8.41 (s, 1H), 7.49 (d,J= 7.4 Hz, 1H), 7.38 - 7.24 (m, 2H), 7.16 (d,J= 7.6 Hz, 1H), 6.08 (bs, 1H), 5.04 (bs, 2H), 4.50 (d,J= 30.8 H<z>, 1H), 4.24 ( d,J= 39.5 Hz, 1H), 4.06 (<s>, 2H), 3.89 (<s>, 1H), 3.37 (d,J= 54.8 Hz, 3H), 2.15 (t,J= 20.8 H<z >, 2H). ESI MS [M+H]+ for C19H24CI2N5O9P2, cal. 598.0, found 598.1

Example 100

Synthesis of [({[(2R,3S,4R,5R))-5-(6-doro-4-{[2-(2-chlorophenyl)etM]amino}-1H-pyrazolo[3,4-d] acid pyrimidin-1-M)-3,4-dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)-phosphoryl)m ethyl]phosphonic

[0413]

[0414] The base compound was synthesized in a similar manner to Example 87.<1>H NMR (400 MHz, DMSO-d e) 68.95 (t,J= 5.6 Hz, 1H), 8.28 - 8.10 (m, 1H), 7.48 - 7.39 (m, 1H), 7.33 (d,J= 6.4 Hz, 1H), 7.29 -7.18 (m, 2H), 6.00 (d,J= 4.0 Hz, 1H), 4.50 (t,J= 4.5 Hz , 1H), 4.25 (t,J= 4.5 Hz, 1H), 4.06 (d,J= 14.9 Hz, 2H), 3.87 (t,J= 5.8 Hz, 1H), 3.69 (q,J= 6.8 Hz, 2H ), 3.10 -3.00 (m, 2H), 2.15 (t,J= 20.4 Hz, 2H). ESI MS [M+H]+ for C19H24CI2N5O9P2, cal. 598.0, found 598.2 Example 101 Synthesis of acid [({[(2R,3S,4R,5R)-5-{4-[benzyl(methyl)amino]-6-chloro-1H-pyrazolo[3,4-d] pyrimidin-1-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)m ethyl]phosphonic

[0415]

[0416] The base compound was synthesized in a similar manner to Example 87. <1>H NMR (400 MHz, DMSO-de) 6 8.35 (s, 1H), 7.30 (dd,J= 20.4, 7.5 Hz, 5H), 6.07 (bs, 1H), 4.99 (bs, 1H), 4.53 (bs, 1H), 4.28 (bs, 1H), 4.05 (<s>, 3H), 3.88 (<s>, 1H), 3.37 3.24 (m , 3H), 2.14 (t,J= 20.9 Hz , 3H). ESI MS [M+H]+ for C19H25CIN5O9P2, cal. 564.1, found 564.1 Example 102 Synthesis of acid [({[(2R,3S,4R,5R)-5-{6-chloro-4-[cyclopentyl(methyl)amino]-1H-pyrazolo[3,4-d] pyrim idin-1-yl}-3,4-dihydroxyoxolan-2-yl]m ethoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0417]

[0418] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MHz, DMSO-d<6>) 6 8.31 (s, 1H), 6.06 (<s>, 1H), 4.51 ( d,J= 4.7 H<z>, 1H), 4.28 (d,J= 5.1 H<z>, 1H), 4.14 -3.96 (m, 2H), 3.88 (<s>, 1H), 3.21 (<s >, 3H), 2.14 (t,J= 19.9 Hz, 2H), 1.67 (bs, 8H). ESI MS [M+H]+ for C17H27CIN5O9P2, cal. 542.1, found 542.2

Example 103

Synthesis of [({[(2R,3S,4R,5R))-5-[6-chloro-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4 acid -dihydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0419]

[0420] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MH<z>, DMSO-d<6>) 6 8.82 (<s>, 1H), 8.16 (s, 1H) , 6.01 (bs, 1H), 4.50 (d,J=5.9 Hz , 1H), 4.26 (bs, 1H), 4.05 (bs, 2H), 3.88 (bs, 1H), 2.95 (d,J=4.6 Hz , 3H), 2.15 (t,J=20.4 Hz, 2H). ESI MS [M+H]+ for C12H19CIN5O9P2, cal. 474.0, found 474.2

Example 104

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1R)-2,2,2-trifluoro-1-phenylethyl]amino}-1H-pyrazolo acid [3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0421]

[0422] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MH<z>, DMSO-d<6>) 69.82 (d,J= 9.3 H<z>, 1H), 8.51 (<s>, 1H), 7.65 (d,J= 7.2 H<z>, 2H), 7.56 - 7.31 (m, 3H), 6.33 (p,J= 8.8 H<z>, 1H), 6.04 (d ,J= 4.2 H<z>, 1H), 4.50 (t,J= 4.4 H<z>, 1H), 4.26 (t,J= 4.6 H<z>, 1H), 4.16 - 4.00 (<m>, 2H), 3.90 (dd,J= 10.6, 5.8 H<z>, 1H), 2.18 (t,J= 20.5 H<z>, 2H).

ESI MS [M-H]" for C19H21CI3N5O9P2, cal. 616.1, found 616.2.

Example 105

Synthesis of acid [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(3S)-oxolan-3-yl]amino}-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)-3,4-dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0423]

[0424] The base compound was synthesized similarly to Example 87.<1>H NMR (400 MH<z>, DMSO-d<6>) 68.95 (d,J= 6.4 Hz, 1H), 8.26 (s, 1H), 6.01 (d,J= 4.2 Hz, 1H), 4.69 -4.59 (m, 1H), 4.50 (t,J= 4.2 Hz, 1H), 4.26 (t,J= 4.5 Hz, 1H), 4.15 - 3.99 (m, 2H), 3.95 - 3.81 (m, 3H), 3.74 (d,J= 7.9 Hz, 1H), 3.67 - 3.58 (m, 1H), 2.35 -2.06 (m, 3H), 1.98 - 1.80 ( m, 1H). ESI MS [M-H]" for C15H22CIN5O10P2, cal. 528.1, found 528.2.

Example 106

Synthesis of [({[(2R,3S,4R,5R))-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4 acid -dih idroxyoxolan-2-yl]methoxy}({[(propan-2-yloxy)carbonyl]oxy}methoxy)phosphoryl)m ethyl]({[(propan-2-yloxy)carbonyl]oxy}m ethoxy)phosphin ico

[0425]

[0426] The base compound was synthesized similarly to Example 69.<1>H NMR (400 MH<z>, DMSO-d<6>) 68.71 - 8.60 (m, 1H), 8.26 -8.15 (m, 1H ), 6.02 -5.96 (m, 1H), 5.60 - 5.38 (m, 5H), 4.87 -4.68 (m, 2H), 4.51 -4.37 (m, 2H), 4.33 - 3.79 (m, 5H), 2.74 -2.53 (m, 2H), 2.07 -1.89 (m, 2H), 1.79 -1.42 (m, 7H), 1.27 -1.12 (m, 12H). ESI MS [M-H]- for C26H40CIN5O15P2, cal. 758.2, found 758.3.

Example 107

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidine acid -1-yl)-3,4-dih idroxyoxolan-2-yl]methoxy}({[(propan-2-yloxy)carbonyl]oxy}-m ethoxy)phosphoryl)m ethyl]({[(propan-2- yloxy)carbonyl]oxy}m ethoxy)phosphinic

[0427]

[0428] The base compound was synthesized in a similar manner to Example 69. <1>H NMR (400 MHz, DMSO-de) 69.17 (d,J= 7.9 Hz, 1H), 8.32 - 8.27 (m, 1H), 7.42 - 7.28 (m, 4H), 7.26 - 7.20 (m, 1H), 6.01 (d,J= 3.7 Hz, 1H), 5.58 - 5.32 (m, 6H), 4.84 -4.69 (m, 2H), 4.49 -4.37 (m, 1H), 4.37 -3.81 (m, 5H), 2.72 -2.52 (m, 1H), 1.53 (d,J= 6.9 Hz, 3H), 1.28 - 1.17 (m, 12H). ESI MS [M-H]" for C29H40CIN5O15P2, cal. 794.2, found 794.2.

Example 108 Synthesis of acid [({[(2R,3R,4S,5R)-5-[4-(benzylamino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-4 -fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0429]

[0430] Step a: 4,6-dieIoro-1H-pyrazoIo[3,4-d]pyrimidine (1.0 g, 5.3 mmol) was dissolved in anhydrous CH3CN (10 mL) and cielopentylamine (478 mg) was added , 5.6 mmol, 1.05 equiv.) followed by TEA (779 pL, 5.6 mmol, 1.05 equiv.). The mixture was stirred at room temperature overnight and then anhydrous CS2CO3 (3.4 g, 10.6 mmol, 2 equiv.) and the bromide (2.2 g, 5.3 mmol) were added. The reaction mixture was stirred at room temperature overnight and then evaporated. The crude residue was dissolved in MeOH (20 mL) and anhydrous K2CO3 (2.2 g, 15.9 mmol, 3 equiv.) was added. The mixture was stirred at room temperature overnight, evaporated with silica gel and purified by column chromatography (SIO2, Hex^- 100% EtOAc) to give first product B (800 mg, 41%) and then product A (600 mg, 30%). For B: ESI Ms [M+H]+ for C15H20CIFN5O3, cal. 372.1, found 372.2.

[0431] Step b: The phosphonylation step was carried out similarly to Example 1 using product B from Step a: <1>H NMR (400 MHz, DMSO-de) 68.76 (d,J= 7.2 Hz , 1H), 8.29 (s, 1H), 6.52 (d,J= 6.5 Hz, 1H), 5.50 - 5.29 (m, 1H), 4.75 (dt,J= 18.7, 7.5 Hz, 1H), 4.43 (h, J= 6.9 Hz, 1H), 4.31 -4.22 (m, 1H), 4.18 -4.05 (m, 1H), 4.04 -3.92 (m, 1H), 2.20 (t,J= 20.5 Hz, 2H), 2.05 - 1.93 (m, 2H), 1.80 - 1.46 (m, 6H). ESI MS [M+H]+ for C16H24CIFN5O8P2, cal. 530.1, found 530.2.

Example 109

Synthesis of [({[(2R,3R,4S,5R))-5-(6-chloro-4-{[(1S}-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidine acid -1-yl}-4-fluoro-3-hydroxyoxolan-2-yljmethoxy}(hydroxy)phosphoryl)-methyl]phosphonic

[0432]

[0433] The base compound was synthesized in a similar manner to Example 108.<1>H NMR (400 MHz, DMSO-d e) 69.19 (d,J= 8.1 Hz, 1H), 8.33 (s, 1H), 7.40 ( d,J=7.9 Hz, 2H), 7.33 (t,J= 7.5 Hz, 2H), 7.23 (t,J= 7.4 Hz, 1H),), 6.34 (dd,J=14.3, 4.6 Hz, 1H), 5.39 (bs, 1H), 5.31 -5.12 (m, 1H), 5.14 (bs, 1H), 4.48 (dt,J= 18.5, 4.5 Hz, 1H), 4.17 (s, 3H), 4.01 (d,J= 5.2 Hz, 2H), 2.24 (t,J= 20.4 Hz, 3H), 1.51 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O8P2, cal. 566.1, found 566.1

Example 110

Synthesis of acid [({[(2R,3R,4S,5R}-5-(6-chloro-4-{[(1R}-1-phenylethyljamino}-1H-pyrazolo[3,4-djpyrimidin-1-yl }-4-fluoro-3-hydroxyoxolan-2-yljm ethoxy}(h idroxy}phosphoryl}-m ethyljphosphonic

[0434]

[0435j The base compound was synthesized similarly to Example 108.<1>H NMR (400 MHz, DMSO-d e) 69.18 (d,J= 8.0 H<z>, 1H), 8.33 (d,J= 1.2 Hz, 1H), 7.40 (d,J= 7.9 Hz, 2H), 7.33 (t,J= 7.3 Hz, 2H), 7.24 (t,J= 7.6 Hz, 1H), 6.50 (d,J= 6.5 Hz, 1H), 5.51 -5.23 (m, 2H), 4.82 -4.66 (m, 1H), 4.22 (bs, 1H), 4.13 -4.02 (m, 1H), 3.94 (bs, 1H), 2.17 (t,J= 20.5 Hz, 2H), 1.53 (d,J= 7.1 Hz, 3H). ESI MS [M+H]+ for C19H24CIFN5O8P2, cal. 566.1, found 566.2

Example 111

Synthesis of [({[(2R,3R,4S,5R))-5-(6-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-N)-4-fluoro-3-hydroxyoxolan-2-yljmethoxy}(hydroxy}-phosphoryl}methyljphosphonic

[0436]

[0437] The base compound was synthesized in a similar manner to Example 108. <1>H NMR (400 MHz, DMSO-d e) 69.27 (d,J= 7.6 Hz, 1H), 8.36 (<s>, 1H), 7.48 -7.40 (<m>, 1H), 7.37 -7.25 (m, 1H), 7.22 - 7.13 (m, 2H), 6.51 (d,J= 6.6 Hz, 1H), 5.59 (p,J= 7.1 Hz, 1H), 5.49 - 5.26 (m, 1H), 4.74 (dt,J= 18.4, 7.6 Hz, 1H), 4.30 -4.17 (m, 1H), 4.15 -4.02 (m , 1H), 3.99 - 3.90 (m , 1H), 2.17 (t,J= 20.5 Hz, 2H), 1.54 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2.

Example 112

Synthesis of [({[(2R,3R,4S,5R))-5-(6-doro-4-{[(1R)-1-(2-f1uorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0438]

[0439] The base compound was synthesized in a similar manner to Example 108. 7.44 (d,J= 8.4 Hz, 1H), 7.29 (t,J= 7.0 Hz, 1H), 7.18 (dt,J= 10.6, 5.6 Hz, 2H), 6.51 (t,J= 4.8 Hz, 1H), 5.60 (t,J= 6.8 Hz, 1H), 5.53 - 5.22 (m, 1H), 4.84 - 4.64 (m, 1H), 4.31 - 4.16 (m, 1H), 4.16 - 4.00 (<m>, 1H), 3.94 (p,J= 3.7 Hz, 1H), 2.18 (t,J= 20.4 Hz, 2H), 1.54 (d,J= 7.2 Hz, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2

Example 113

Synthesis of [({[(2R,3R,4S,5R))-5-(6-chloro-4-{[(1S)-1-(3-f1uorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0440]

[0441] The base compound was synthesized in a similar manner to Example 108.<1>H NMR (400 MHz, DMSO-d e) 69.21 (d,J= 7.8 Hz, 1H), 8.33 (s, 1H), 7.46 - 7.30 (m, 1H), 7.27 -7.16 (m, 2H), 7.12 -7.00 (m, 1H), 6.51 (d,J= 6.5 Hz, 1H), 5.49 -5.26 (m, 2H), 4.74 (dt, J= 18.4, 7.6 Hz, 1H), 4.29 -4.18 (m, 1H), 4.14 -4.02 (<m>, 1H), 4.00 - 3.89 (m, 1H), 2.17 (t,J= 20.5 H<z> , 2H), 1.54 (d,J= 7.0 Hz , 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2.

Example 114

Synthesis of [({[(2R,3R,4S,5R))-5-(6-doro-4-{[(1R)-1-(2-f1uorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0442]

[0443] The base compound was synthesized in a similar manner to Example 108. <1>H NMR (400 MHz, DMSO-d e) 69.21 (d,J= 8.0 H<z>, 1H), 8.34 (<s>, 1H), 7.42 -7.32 (m, 1H), 7.27 -7.20 (<m>, 2H), 7.12 -7.03 (<m>, 1H), 6.51 (d,J= 6.5 Hz, 1H), 5.49 -5.29 ( m, 2H), 4.74 (dt,J= 18.8, 7.8 Hz, 1H), 4.29 - 4.18 (m, 1H), 4.15 - 4.03 (<m>, 1H), 3.99 - 3.90 (<m>, 1H), 2.17 (t,J= 20.5 H<z>, 2H), 1.54 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2.

Example 115

Synthesis of [({[(2R,3R,4S,5R))-5-(6-chloro-4-{[(1S}-1-(4-fluorophenyl}ethyl]amino}-1H-pyrazolo acid[3, 4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxyl(hydroxy)-phosphoryl)methyl]phosphonic

[0444]

[0445] The base compound was synthesized similarly to Example 108.<1>H NMR (400 MHz, DMSO-d e) 69.22 (d,J= 7.9 Hz, 1H), 8.34 (s, 1H), 7.45 ( dd,J= 8.7, 5.6 Hz, 2H), 7.17 (t,J= 8.9 Hz, 2H), 6.52 (d,J= 6.5 Hz, 1H), 5.53 - 5.27 (m, 2H), 4.75 (dt,J = 18.7, 7.6 Hz, 1H), 4.31 -4.20 (m , 1H), 4.17 -4.04 (m , 1H), 3.97 (dd,J= 7.5, 3.9 Hz, 1H), 2.19 (t,J= 20.5 Hz , 2H), 1.54 (d,J= 7.0 Hz, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2

Example 116

Synthesis of [({[(2R,3R,4S,5R))-5-(6-doro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)-4-f1uoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)fbsphoryl)methyl]phosphonic

[0446]

[0447] The base compound was synthesized in a similar manner to Example 108. <1>H NMR (400 MHz, DMSO-d e) 69.21 (d,J= 7.9 Hz, 1H), 8.34 (<s>, 1H), 7.54 -7.37 (m, 2H), 7.17 (t,J= 8.8 Hz, 2H), 6.52 (d,J= 6.6 Hz, 1H), 5.60 - 5.23 (m, 2H), 4.76 (dt,J= 18.7, 7.6 Hz, 1H), 4.24 (dt,J= 7.4, 4.8 Hz, 1H), 4.09 (dt,J= 10.9, 7.4 Hz, 1H), 3.96 (dt,J= 10.7, 5.3 Hz, 1H), 2.19 ( t,J= 20.5 Hz, 2H), 1.54 (d,J= 7.1 Hz, 3H). ESI MS [M+H]+ for C19H23CIF2N5O8P2, cal. 584.1, found 584.2 Reference Example 117

Synthesis of acid [({[(2R,3S,4R,5R))-5-[5-chloro-7-(cyclopentylamino)-3H-imidazo[4,5-b]pyridin-3-yl]-3,4 -dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic

[0448]

[0449] added

N,0-Bis(trimethylsi\yl)acetamide (0.523 mL, 2.14 mmol) dropwise and the reaction mixture was heated to 85 °C for 1 hour. The mixture was cooled to r.t. and a solution of beta-D-ribofuranose 1,2,3,5-tetraacetate (726 mg, 2.28 mmol) in MeCN (7 mL) and trimethylsilyl trifluoromethanesulfonate (0.471 mL, 2.60 mmol) were added dropwise sequentially. mmol). The reaction mixture was heated to 85 °C for 4 hours. The mixture was cooled and saturated aqueous sodium bicarbonate (50 mL) was added, subsequently extracted three times with EtOAc (100 mL), dried over sodium sulfate and concentrated.

[0450] Step b: Dioxane (2 mL) and cyclopentylamine (0.987 mL, 10 mmol) were added to the residue. The mixture was heated at 100 °C for 16 hours. The reaction mixture was loaded on silica gel and purified by silica gel chromatography (0-10% MeOH in DCM) to obtain the desired product as a brown solid (298 mg, 40%).

[0451] Step c: The base compound was synthesized as a white solid (10 mg; 6%) similarly to Example 1.1H NMR (400 MH<z>, DMSO-d6) 68.40 (<s>, 1H), 7.12 (d,J=7.3 H<z>, 1H), 6.40 (<s>, 1H), 5.91 (d,J= 5.8 H<z>, 1H), 4.53 (t,J= 5.4 Hz, 1H) , 4.23 -4.18 (m, 1H), 4.18 - 4.05 (m, 3H), 2.26 (t,J= 20.5 Hz , 2H), 2.04 -1.91 (m, 2H), 1.76 - 1.64 (m, 2H), 1.64 -1.48 (m, 4H). ESI MS [M-H]- for C17H24CIN4O9P2, cal. 525.1, found 525.2

Reference Example 118

Synthesis of [({[(2R,3R,4S,5R))-5-[5-chloro-7-(cyclopentytamino)-3H-imidazo[4,5-b]pyridin-3-yl]-4-fluoro acid -3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0452]

[0453] Step a: To a solution of 5,7-didoro¡m¡dazo[4,5-b]pyrádine (564 mg, 3 mmol) in MeCN (18 mL) at r.t. sodium hydride (130 mg, 3.24 mmol, 60% suspension in oil) was added. The reaction mixture was stirred at r.t. for 30 minutes. A solution of 2,3,5-Tri-O-benzoyl-D-ribofuranosyl bromide in MeCN (4 mL) was added at r.t. and the reaction mixture was stirred at r.t. for 14 hours. The reaction mixture was quenched by adding methanol (5 mL) and sodium bicarbonate (5 g), filtered through celite and concentrated.

[0454] Step b: 1) Dioxane (5 mL) and cyclopentylamine (1.48 mL, 15 mmol) were added to the residue. The mixture was heated at 100 °C for 20 hours. The reaction mixture was cooled to rt.

[0455] 2) Potassium carbonate (4 g) and methanol (20 mL) were added at r.t. and the reaction mixture was stirred at r.t. for 1 hour. Excess solvent was removed in vacuo and the crude residue was purified by silica gel chromatography (0-15% MeOH in DCM) to obtain the desired product as a brown solid (499 mg, 45%).

[0456] Step c: The base compound was synthesized as a white solid (26 mg; 10%) in a similar manner to Example 1. <1>H NMR (400 MH<z>, DMSO-d<6>) 68.24 ( d,J= 2.3 H<z>, 1H), 7.19 (br<s>, 1H), 6.42 (dd,J= 15.4, 4.4 H<z>, 1H), 6.42 (<s>, 1H), 5.23 (dt,J= 52.4, 4.1 Hz, 1H), 4.58 -4.44 (m, 1H), 4.19 (t,J= 6.1 Hz, 2H), 4.08 -3.99 (m, 1H), 2.27 (t,J= 20.5 Hz , 2H), 2.04 - 1.90 (m, 2H), 1.78 - 1.64 (m, 2H), 1.64 - 1.47 (m, 4H). ESI MS [M-H]- for C17H23CIFN4O8P2, cal. 527.1, found 527.2.

Example 119

Synthesis of [({[(2R,3S,4R,5R))-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-b]pmdin-1-yl]-3,4 acid -d¡hydroxy¡oxolan-2-¡l]methoxy¡}(h¡drox¡)phosphor¡l)methyl]-phosphon¡co

[0457]

reflux

Stage f

.

[0458] Step a: Ethyl (ethoxymethylen)cyanoacetate (50.5 g, 299.0 mmol) was dissolved in anhydrous EtOH (350 mL) and then product hydrazine (50 g, 328.9 mmol, 1.1 equiv.). The reaction mixture was stirred at reflux overnight and then evaporated. The solid residue was washed with MTBE to give a white solid (55.5 g, 63%). ESI MS [M+H]+ for C14H18N3O3, cal. 276.1, found 276.2.

[0459] Step b: Diethyl malonate (90 mL, 0.59 mol, 4 equiv.) was dissolved in anhydrous EtOH (300 mL) and cooled to 0° C (ice bath). A 21% solution of NaOEt in EtOH (220 mL, 0.59 mol, 4 equiv.) was added dropwise (over 10 min), then the cooling bath was removed and the reaction was stirred at room temperature for 15 min. The solid product from Step a (40.4 g, 147 mmol) was added portionwise (over 2 min) and the reaction mixture was stirred at reflux for 5 days, then evaporated. The residue was diluted with H2O (1.2 L) and neutralized to pH~5 with A<c>OH. The product was filtered, washed with H2O (200 mL) and dried under vacuum (48.4 g, 96%). ESI<m>S [M+H]+ for C17H18N3O5, cal. 344.1, found 344.2.

[0460] Step c: The product from step b (48.4 g, 141.1 mmol) was dissolved in 15% aqueous NaOH (500 mL) and stirred at reflux for 5 h. Cooled to 0° C and carefully neutralized with AcOH to pH~5. The white solid was filtered, washed with H2O (100 mL) and dried under vacuum (38 g, euant.). ESI MS [M+H]+ for C14H14N3O3, cal. 272.1, found 272.2.

[0461] Step d: The mixture of the product from step c (38 g, 140.2 mmol) and phenylphosphonium dichloride (79.5 mL, 560.8 mmol, 4 equiv.) was stirred at 170 ° C for 7 h , then cooled to -80° C and poured onto vigorously shaken ice. A brown, sticky material precipitated and, after prolonged stirring, turned into a solid. The cooled mixture was neutralized with concentrated aqueous NH3 to pH~7 and the product was extracted with CH2Cl2 (2 * 400 mL). The combined organics were dried over MgSO4, filtered and evaporated to give a product which was used without further purification (24 g, 55%). ESI MS [M+H]+ for C14H12CI2N3O, cal. 308.0, found 308.1.

[0462] Step e: The product from step d (22 g, 71.4 mmol) was dissolved in TFA (75 mL) and stirred at 60 ° C for 12 h, then cooled and poured into H2O (600 mL). The gray solid was filtered, washed with saturated NaHC03, then H2O and dried under vacuum. ESI MS [M+H]+ for C6H4CI2N3, cal. 188.0, found 188.1.

[0463] Step f: The product of step f was synthesized similarly to Example 87.1H NMR (400 MHz, DMSO-d 6) 6 8.55 (s, 1H), 7.72 (s, 1H), 6.48 (d, J= 3.0 Hz , 1H), 5.90 - 5.83 (m , 1H), 5.67 - 5.61 (m , 1H), 4.46 - 4.38 (m , 1H), 4.33 (ddd,J= 12.1, 3.5, 1.2 H<z> , 1H), 4.05 (ddd,J= 12.2, 5.1, 1.2 H<z>, 1H), 2.09 (<s>, 3H), 2.06 (<s>, 3H), 1.96 (<s>, 3H). ESI MS [M+H]+ for C17H18CI2N3O7, cal. 446.0, found 446.1.

[0464] Step g: The product of step g was synthesized in a similar manner to Example 87. ESI MS [M+H]+ for C16H22CIN4O4, cal. 369.1, found 369.2.

[0465] Step h: The base compound was synthesized in a similar manner to Example 87. <1>H NMR (400 MHz, DMSO-d<6>) 68.27 (<s>, 1H), 7.66 (d,J= 6.7 H<z>, 1H), 6.22 (<s>, 1H), 6.08 (d,J= 4.2 H<z>, 1H), 4.51 (t,J= 4.7 H<z>, 1H), 4.26 (t ,J= 5.1 H<z>, 1H), 4.17 -3.83 (m , 4H), 2.17 (t,J= 20.5 Hz , 2H), 2.06 -1.92 (m , 2H), 1.77 - 1.45 (m , 6H) . ESI MS [M+H]+ for C17H26CIN4O9P2, cal. 527.1, found 527.2.

Example 120

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridine acid -1-yl}-3,4-dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]-phosphonic

[0466]

[0467] The base compound was synthesized in a similar manner to Example 119. H NMR (400 MH<z>, DMSO-d<6>) 68.38 (<s>, 1H), 8.20 (d,J= 7.2 Hz, 1H ), 7.42 - 7.36 (m , 2H), 7.35 -7.27 (m , 2H), 7.24 - 7.18 (m , 1H), 6.08 - 5.97 (m , 2H), 4.85 (s, 1H), 4.50 (t,J = 4.5 H<z>, 1H), 4.25 (t,J= 4.8 H<z>, 1H), 4.14 - 3.97 (<m>, 2H), 3.93 - 3.81 (<m>, 1H), 2.17 (t ,J= 20.5 H<z>, 2H), 1.52 (d,J= 6.2 Hz , 3H). ESI MS [M+H]+ for C20H26CIN4O9P2, cal. 563.1, found 563.2.

Example 121

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridine acid -1-yl)-3,4-dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]-phosphonic

[0468]

[0469] The base compound was synthesized in a similar manner to Example 119. <1>H NMR (400 MH<z>, DMSO-d<6>) 68.38 (<s>, 1H), 8.20 (d,J= 7.2 Hz , 1H), 7.44 - 7.35 (m , 2H), 7.35 -7.28 (m , 2H), 7.25 - 7.17 (m , 1H), 6.12 - 5.93 (m , 2H), 4.85 (s, 1H), 4.57 - 4.48 (m , 1H), 4.25 (t,J= 4.9 Hz , 1H), 4.12 -3.95 (m , 2H), 3.91 -3.79 (m , 1H), 2.17 (t,J=20.5 Hz , 2H), 1.51 (d,J= 6.6 Hz, 3H). ESI MS [M+H]+ for C20H26C1N4O9P2, cal. 563.1, found 563.2.

Example 122

Synthesis of acid [({[(2R,3S,4R,5R}-5-(6-doro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0470]

[0471] The base compound was synthesized in a similar manner to Example 119.<1>H NMR (400 MHz, DMSO-d e) 68.38 (s, 1H), 8.23 (d,J= 6.9 Hz, 1H), 7.42 - 7.34 (m, 1H), 7.33 - 7.09 (m, 3H), 6.06 (d,J= 4.3 Hz, 1H), 5.97 (s, 1H), 5.04 (s, 1H), 4.53 -4.47 (m, 1H) , 4.25 (t,J= 4.7 Hz, 1H), 4.13 -3.97 (m, 2H), 3.92 -3.82 (m, 1H), 2.16 (d,J= 20.5 H<z>, 2H), 1.56 (d, J= 6.4 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O9P2, cal. 581.1, found 581.2.

Example 123

Synthesis of [({[(2R,3S,4R,5R))-5-(6-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3, 4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0472]

[0473] The base compound was synthesized similarly to Example 119.<1>H NMR (400 MHz, DMSO-d e) 68.36 (s, 1H), 8.18 (d,J=7.2 Hz, 1H), 7.46 - 7.39 (m, 2H), 7.19 -7.10 (m, 2H), 6.13 -5.99 (m, 2H), 4.89 (s, 1H), 4.53 -4.46 (m, 1H), 4.25 (t,J=4.8 Hz, 1H), 4.12 - 3.97 (m, 2H), 3.92 - 3.81 (m, 1H), 2.18 (t,J= 20.5 Hz, 2H), 1.50 (d,J= 7.3 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O9P2, cal. 581.1, found 581.2.

Example 124

Synthesis of [({[(2R,3R,4S,5R))-5-[6-chloro-4-(dclopentylamino)-1H-pyrazolo[3,4-b]pyridin-1-yl]-4-fluoro acid -3-h idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0474]

m well

[0475] Step a: To the mixture of 4,6-didoro-1H-pyrazolo[3,4-b]pyridine (2.1 g, 11.1 mmol) and the bromide (4.7 g, 11.1 mmol) in anhydrous CH3CN (50 mL), CS2CO3 (4.3 g, 13.3 mmol, 1.2 equiv.) was added and the reaction mixture was stirred at room temperature overnight. Evaporated with silica gel and purified by column chromatography (SÍO2, Hex^-Hex:EtOAc, 2:8) to give a white solid (2.5 g, 42%).<1>H NMR (400 MH <z>, DMSO-d<6>) 68.53 (<s>, 1H), 8.05 (d,J= 8.4 Hz, 2H), 7.91 (d,J= 8.4 Hz, 2H), 7.75 - 7.65 (m, 2H), 7.65 - 7.51 (m, 3H), 7.49 - 7.41 (m, 2H), 6.96 (d,J= 6.6 Hz, 1H), 6.45 (dt,J= 15.8, 7.2 Hz, 1H), 6.19 - 5.97 (m, 1H), 4.78 - 4.53 (m, 3H). ESI MS [M+H]+ for C25H19CI2FN3O5, cal. 530.1, found 530.2.

[0476] Step b: The mixture of the product from step a (500 mg, 0.94 mmol), cielopentylamine (84 mg, 0.99 mmol, 1.05 equiv.), TEA (138 pL, 0.99 mmol , 1.05 equiv.) in anhydrous EtOH (5 mL) was placed in a pressurized vial and heated at 110 °C for 2 days. Cooled to room temperature and K2CO3 (262 mg, 1.9 mmol, 2 equiv.) was added and the reaction mixture was stirred overnight. It was evaporated with silica gel and purified by column chromatography (SÍO2, H e x ^ 100% EtOAe) to give a white solid (170 mg, 49%).<1>H NMR (400 MHz, DMSO-d<6 >) 68.28 (s, 1H), 7.66 (d,J= 6.9 Hz, 1H), 6.53 (d,J= 6.6 Hz, 1H), 6.23 (<s>, 1H), 5.80 (d,J= 5.7 Hz , 1H), 5.45 -5.23 (m, 1H), 4.82 -4.61 (m, 2H), 3.98 (s, 1H), 3.80 - 3.50 (m , 3H), 2.09 - 1.89 (m, 2H), 1.76 - 1.46 (m, 6H). ESI MS [M+H]+ for C16H21CIFN4O3, cal. 371.1, found 371.3.

[0477] Step c: The base compound was synthesized in a similar manner to Example 1. <1>H NMR (400 MHz, DMSO-d<6>) 68.30 (s, 1H), 7.69 (d,J= 6.7 Hz, 1H), 6.55 (d,J= 6.6 Hz, 1H), 6.23 (s, 1H), 5.48 -5.25 (m, 1H), 4.77 (dt,J= 18.1,7.6 Hz, 1H), 4.28 - 4.18 (m , 1H), 4.13 - 3.88 (m, 3H), 2.17 (t,J= 20.5 Hz , 2H), 2.07 - 1.93 (m, 2H), 1.77 - 1.44 (m, 6H). ES/MS [M+H]+ for C17H25C1FN4O8P2, cal. 529.1, found 529.1.

Example 125

Synthesis of acid [({[(2R,3R,4S,5R}-5-(6-chloro-4-{[(1S}-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridin) -1-yl}-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0478]

[0479] The base compound was synthesized similarly to Example 124.<1>H NMR (400 MH<z>, DMSO-d<6>) 68.40 (<s>, 1H), 8.23 (d,J= 7.1 Hz, 1H), 7.44 - 7.37 (m, 2H), 7.37 - 7.28 (m, 2H), 7.26 -7.16 (m, 1H), 6.54 (d,J= 6.5 Hz, 1H), 6.04 (<s>, 1H), 5.36 (dt,J= 53.5, 7.1 Hz, 1H), 4.93 -4.67 (m, 2H), 4.27 -4.19 (m, 1H), 4.14 -4.02 (m , 1H), 3.98 -3.85 (m, 1H), 2.17 (t,J= 20.5 Hz, 2H), 1.52 (d,J= 6.4 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O8P2, cal. 565.1, found 565.2.

Example 126

Synthesis of [({[(2R,3R,4S,5R))-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridine acid -1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0480]

[0481] The base compound was synthesized similarly to Example 124. <1>H NMR (400 MHz, DMSO-d<6>) 68.41 (s, 1H), 8.22 (d,J= 7.1 Hz, 1H), 7.43 - 7.36 (m, 2H), 7.36 - 7.27 (m, 2H), 7.26 -7.17 (m, 1H), 6.54 (d,J= 6.5 Hz, 1H), 6.04 (<s>, 1H), 5.37 ( dt,J= 53.7, 7.2 Hz, 1H), 4.96 - 4.67 (m, 2H), 4.27 -4.18 (m, 1H), 4.13 - 4.01 (<m>, 1H), 3.97 - 3.87 (m, 1H), 2.18 (t,J= 20.5 Hz, 2H), 1.52 (d,J= 6.4 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O8P2, cal. 565.1, found 565.2.

Reference Example 127

Synthesis of acid [({[(2R,3S,4R,5R)-5-(2-doro-4-{[(1S)-1-(2-fluoro1phenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0482]

Stage c

[0483] Step a: To the mixture of 2,4-didoro-7H-pyrroIo[2,3-d]pyrnidine (14.8 g, 78.9 mmol) and the bromide (40 g, 118.3 mmol, 1.5 equiv.) in anhydrous CH3CN (600 mL), CS2CO3 (38.6 g, 118.3 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at room temperature overnight. Evaporated with silica gel and purified by column chromatography (SIO2, H e x ^ Hex:EtOAc, 2:8) to give a white solid (13.8 g, 39%). ESI m S [M+H]+ for C17H18CI2N3O7, cal.

446.0, found 446.1.

[0484] Steps b and c were performed similarly to Example 1. <1>H NMR (400 MHz, DMSO-d<6>) 68.38 (d,J= 7.9 Hz, 1H), 7.47 -7.36 (m, 2H ), 7.32 -7.23 (m, 1H), 7.20 -7.09 (m, 2H), 6.81 (s, 1H), 5.96 (d,J= 6.0 Hz, 1H), 5.58 (t,J= 7.3 Hz, 1H) , 4.29 (t,J= 5.7 Hz, 1H), 4.14 -3.96 (m, 4H), 2.24 (t,J= 20.5 Hz, 2H), 1.51 (d,J= 6.9 Hz, 3H). ESI MS [M+H]+ for C20H25C1FN4O9P2, cal. 581.1, found 581.2.

Reference Example 128

Synthesis of acid [({[(2R,3S,4R,5R)-5-(2-chloro-4-{[(1S)-1-(3-fluorophenN)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-N)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-m ethyl]phosphonic

[0485]

[0486] The base compound was synthesized in a similar manner to Example 127. <1>H NMR (400 MHz, DMSO-d e) 68.34 (d,J= 8.1 Hz, 1H), 7.42 - 7.30 (m, 2H), 7.26 - 7.16 (m, 2H), 7.08 - 6.98 (m, 1H), 6.77 (s, 1H), 5.97 (d,J= 6.0 Hz, 1H), 5.45 - 5.33 (m, 1H), 4.29 (t, J= 5.5 Hz, 1H), 4.14 -3.98 (m, 4H), 2.24 (d,J= 20.5 Hz, 2H), 1.51 (d,J= 6.8 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O9P2, cal. 581.1, found 581.2.

Reference Example 129

Synthesis of acid [({[(2R,3S,4R,5R))-5-(2-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)-phosphoryl)m ethyl]phosphonic

[0487]

[0488] The base compound was synthesized similarly to Example 127.<1>H NMR (400 MHz, DMSO-d e) 68.32 (d,J= 8.0 H<z>, 1H), 7.46 -7.34 (m, 3H), 7.17 -7.08 (<m>, 2H), 6.76 (<s>, 1H), 5.97 (d,J= 6.3 Hz, 1H), 5.44 -5.33 (m, 1H), 4.29 (t,J= 5.8 Hz, 1H), 4.14 -3.97 (m, 4H), 2.24 (d,J= 20.5 Hz, 2H), 1.50 (d,J= 6.9 Hz, 3H). ESI MS [M+H]+ for C20H25CIFN4O9P2, cal. 581.1, found 581.2.

Reference Example 130

Synthesis of [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1R)-1-phenylethyl]amino}-7H-pyrrolo[2,3-d]pyrimidine acid -7-yl)-4-fluoro-3-h idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0489]

[0490] The base compound was synthesized in a similar manner to Example 65. <1>H NMR (400 MHz, DMSO-d e) 68.36 (d,J= 8.4 Hz, 1 H), 7.39 (d,J= 7.6 Hz , 2 H), 7.35 -7.23 (m, 3 H), 7.21 (t,J= 7.2 Hz, 1 H), 6.78 (s, 1 H), 6.44 (dd,J= 15.5, 4.6 Hz, 1 H) , 5.40 (t,J= 8.1 Hz, 1 H), 4.40 (dt,J= 18.8, 4.6 Hz, 1 H), 4.13 (d,J= 6.7 Hz, 2 H), 3.95 (q,J= 5.1 Hz , 1 H), 2.22 (t,J= 20.3 Hz , 2 H), 1.51 (d,J= 7.0 Hz, 3 H). ESI MS [M-H]- for C209H23CIFN4O8P2, cal. 563.1, found 563.2.

Reference Example 131

Synthesis of [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1S)-1-phenylethyl]amino}-7H-pyrrolo[2,3-d]pyrimidine acid -7-yl)-4-fluoro-3-h idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)-m ethyl]phosphonic

[0491]

[0492] The base compound was synthesized in a similar manner to Example 65. <1>H NMR (400 MHz, DMSO-d e) 68.37 (d,J= 8.3 Hz, 1 H), 7.39 (d,J= 7.7 Hz , 2 H), 7.31 (t,J= 7.5 Hz, 2 H), 7.28 - 7.16 (m, 2 H), 6.79 (s, 1 H), 6.44 (dd,J= 15.8, 4.5 Hz, 1 H) , 5.39 (<s>, 1 H), 4.39 (dt,J= 18.7, 4.4 Hz, 1 H), 4.12 (m, 2 H), 3.95 (q,J= 5.1 Hz, 1 H), 2.23 (t ,J= 20.5 H<z>, 2 H), 1.51 (d,J= 7.0 Hz , 3 H). ESI MS [M+H]+ for C20H25CIFN4O8P2, cal. 565.1, found 565.2.

Reference Example 132

Synthesis of [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1S)-1-(3-fluoro1phenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0493]

[0494] The base compound was synthesized similarly to Example 65.<1>H NMR (400 MHz, DMSO-d e) 68.40 (d,J= 8.0 Hz, 1 H), 7.40 -7.16 (m, 4 H ), 7.03 (td,J= 8.7, 2.6 Hz, 1 H), 6.78 (d,J= 3.9 Hz, 1 H), 6.45 (dd,J= 15.7, 4.4 Hz, 1 H), 5.43 -5.34 (m , 1 H), 5.11 (dt,J= 52.7, 4.0 Hz , 1 H), 4.38 (dq,J= 18.7, 4.5 Hz, 1 H), 4.18 -4.06 (m , 1 H), 3.96 (q,J = 5.0 Hz, 1 H), 2.25 (t,J= 20.5 Hz, 2 H), 1.51 (d,J= 6.9 Hz, 3 H). ESI MS [M-H]- for C20H22CIF2N4O8P2, cal. 581.1, found 581.2.

Reference Example 133

Synthesis of acid [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0495]

[0496] The base compound was synthesized in a similar manner to Example 65.<1>H NMR (400 MHz, DMSO-d e) 68.37 (d,J= 8.1 H<z>, 1 H), 7.42 (dd,J = 8.4, 5.5 Hz, 2 H), 7.26 (t,J= 3.1 Hz, 1 H), 7.22 - 7.09 (m, 2 H), 6.77 (d,J= 3.6 Hz, 1 H), 6.44 (dd, J=15.5, 4.5 Hz, 1 H), 5.43 -5.34 (m, 1 H), 5.14 (dt,J= 52.7, 4.0 H<z>, 1 H), 4.40 (dt,J= 18.7, 4.3 Hz, 1 H), 4.20 -4.02 (m, 2 H), 3.95 (q,J= 5.1 Hz, 1 H), 2.24 (t,J=20.5 H<z>, 2 H), 1.50 (d,J= 7.0 Hz, 3H). ESI MS [M-H]- for C20H22CIF2N4O8P2, cal. 581.1, found 581.2.

Reference Example 134

Synthesis of acid [({[(2R,3R,4S,5R)-5-(2-chloro-4-[(1S)-1-(4-fluorophenyl)ethyl]aminol-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-M]methoxy}(hydroxy)-phosphoryl)metM]phosphonic

[0497]

[0498] The base compound was synthesized in a similar manner to Example 65. <1>H NMR (400 MHz, DMSO-d e) 68.38 (d,J= 7.7 Hz, 1 H), 7.41 (dd,J= 8.6, 5.5 Hz, 2 H), 7.26 (<s>, 1 H), 7.18 -7.08 (<m>, 2 H), 6.77 (<s>, 1 H), 6.44 (dd,J= 15.6, 4.3 Hz, 1 H), 5.38 (s, 1 H), 5.24 -4.96 (m, 1 H), 4.45 -4.34 (m, 1 H), 4.13 (s, 2 H), 3.95 (d,J= 5.2 Hz, 1 H), 2.23 (t,J= 20.4 Hz, 2 H), 1.50 (d,J= 7.1 Hz, 3 H).ESI MS [M+H]+ for C20H24CIF2N4O8P2, cal. 583.1, found 583.2.

Reference Example 135

Synthesis of [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(hydroxy)-phosphoryl)methyl]phosphonic

[0499]

[0500] The base compound was synthesized similarly to Example 65. <1>H NMR (400 MHz, DMSO-de) 68.46 (d, J = 7.8 Hz, 1 H), 7.44 (t, J = 7.8 Hz, 1 H), 7.28 (dt, J = 11.3, 4.5 Hz, 2H), 7.17 (q, J = 8.1,7.4 Hz, 2 H), 6.83 (d, J = 3.9 Hz, 1 H), 6.47 (dd, J = 15.7, 4.5 Hz, 1 H), 5.60 (t, J = 7.4 Hz, 1 H), 5.13 (dt, J = 52.5, 4.2 Hz, 1 H), 4.41 (dt, J = 18.8, 4.4 Hz, 1 H), 4.14 (td, J = 12.0, 10.7, 5.7 Hz, 2 H), 3.97 (q, J = 5.0 H<z>, 1 H), 2.27 (t, J = 20.5 H<z>, 2 H), 1.53 (d, J = 7.0 Hz, 3 H). ESI MS [M+H]+ for C20H24CIF2N4O8P2, cal. 583.1, found 583.2.

Reference Example 136

Synthesis of acid [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-M]methoxy}(hydroxy)-phosphoryl)metM]phosphonic

[0501]

[0502] The base compound was synthesized in a similar manner to Example 65. <1>H NMR (400 MHz, DMSO-d e) 68.44 (s, 1 H), 7.43 (t,J= 7.8 Hz, 1 H), 7.28 (s, 2 H), 7.21 -7.09 (m, 2 H), 6.44 (dd,J= 15.8, 4.7 Hz, 1 H), 5.59 (s, 1 H), 5.14 (d,J= 52.8 Hz, 1 H), 4.40 (d,J= 18.9 Hz, 1 H), 4.13 (<s>, 2 H), 3.95 (d,J= 5.7 Hz, 1 H), 2.24 (t,J= 20.7 H<z >, 2 H), 1.51 (d,J= 6.9 Hz, 3 H). e S i MS [M+H]+ for C20H24CIF2N4O8P2, cal. 583.1, found 583.2.

Reference Example 137

Synthesis of [({[(2R,3R,4S,5R))-5-(2-chloro-4-{[(1R)-1-(3-fluorophenyl)ethyl]amino}-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)-phosphoryl)m ethyl]phosphonic

[0503]

[0504] The base compound was synthesized in a similar manner to Example 65. <1>H NMR (400 MHz, DMSO-d e) 68.39 (d,J= 8.1 Hz, 1 H), 7.35 (td,J= 8.0, 7.5, 6.0 Hz , 1 H), 7.31 -7.17 (m, 3 H), 7.04 (td,J= 8.6 , 2.5 Hz, 1 H), 6.81 -6.75 (m, 1 H), 6.45 (dd,J= 15.6, 4.4 Hz, 1 H), 5.44 -5.35 (m, 1 H), 5.14 (dt,J= 52.8, 4.1 Hz, 1 H), 4.40 (dt,J= 18.8, 4.4 Hz, 1 H), 4.19 -4.09 (m, 1 H), 3.95 (q,J= 5.0 Hz, 1 H), 2.24 (t,J= 20.5 Hz, 2 H), 1.50 (d,J= 6.9 Hz, 3 H). ESI MS [M-H]- for C20H22CIF2N4O8P2, cal. 581.1, found 581.2.

Reference Example 138

Synthesis of acid [({[(2R,3S,4R,5R))-5-[6-chloro-4-(cyclopentylamino)-1H-imidazo[4,5-c]pyridin-1-yl]-3,4 -dih idroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0505]

Stage a

0<3>

for room temperature

[0506] Step a: To a solution of p-D-ribofuranose-1,2,3,5-tetraaeetate (4.07 g, 12.8 mmol) and 4,6-dieIoro-1H-imidazo[4,5-c ]pyridine (2.0 g, 10.6 mmol) in ACN (64 mL) TMSOTf (4.6 mL, 25.6 mmol) was added via Syringe. Subsequently, DBU (1.9 mL, 12.8 mmol) was added dropwise and the reaction was stirred at room temperature for 2 hours. The reaction was cooled to 0°C and poured into cold saturated NaHCOa solution. This mixture was transferred to a separatory funnel and extracted with DCM (3x). The combined organics were dried over MgS0<4>and concentrated to dryness. The crude material (1.4 g) was used without further purification. ESI MS [M+H]+ for C17H17CI2N3O7, cal. 446.0, found 446.1.

[0507] Step b: To a flask with a rosette stopper containing the crude dichloride (1.4 g), ethylopentylamine (7 mL) was added. The vial was sealed and heated to 80°C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was reconstituted in DCM and purified by column chromatography (SO 2.0 to 15% MeOH/DCM) to obtain the desired product (352 mg). ESI m S [M+H]+ for C16H21CIN4O4, cal. 368.1, found 369.2.

[0508] Step c: The base compound was synthesized in a similar manner to Example 1. <1>H NMR (400 MHz, DMSO-d<6>) 68.33 (<s>, 1H), 7.05 (<s>, 1H ), 6.88 (<s>, 1H), 5.77 (d,J= 5.9 H<z>, 1H), 4.43 (br.<s>, 1H), 4.28 (dd,J= 5.9, 5.0 H<z> , 1H), 4.21 - 4.04 (m, 5H), 2.28 (t,J= 20.5 Hz , 2H), 2.05 - 1.77 (m, 2H), 1.79 - 1.45 (m, 7H). ESI MS [M-H]- for C17H25CIN4O9P2, cal. 525.1, found 525.2.

Reference Example 139

Synthesis of [({[(2R,3R,4S,5R))-5-[6-chloro-4-(cyclopentylamino)-1H-imidazo[4,5-c]pyridin-1-yl]-4-fluoro acid -3-hydroxyoxolan-2-yl]m ethoxy}(h idroxy)phosphoryl)m ethyl]phosphonic

[0509]

Stage a

0 C for room temperature

[0510] Step a: A solution of 4,6-didoro-1H-imidazo[4,5-c]pyridine (2.0 g, 10.6 mmol) and 2-Deoxy-2-fIuoro--D bromide -arabinofuranosyl 3,5-dibenzoate (4.95 g, 11.7 mmol; Ca S: 97614-44-3) in 50 mL acetonitrile was treated with CS2CO3 (4.16 g, 12.8 mmol). The mixture was allowed to stir for 3 hours at room temperature, then diluted with ethyl acetate and washed with water and brine. The organics were dried over MgSO4 and concentrated under reduced pressure. The crude product obtained was used without further purification. ESI MS [M+H]+for C25H18CI2FN3O5, cal. 530.1, found 530.2.

[0511] Step b: To a rose-stoppered flask containing the crude dichloride (3.5 g, 6.6 mmol) was added cielopentylamine (18 mL). The vial was sealed and heated to 80°C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was reconstituted in DCM and purified by column chromatography (SÍO2, 0 to 15% MeOH/DCM) to obtain the desired product. ESI MS [M+H]+ for C16H20CIFN4O3, cal. 371.1, found 371.2.

[0512] Step<c>: The title compound was synthesized similarly to example 1: 1H NMR (400 MH<z>, DMSO-d 6) 68.22 (d,J= 1.8 Hz, 1H), 7.06 (< s>, 2H), 6.98 -6.82 (m, 1H), 6.37 (dd,J=15.9, 4.4 Hz, 1H), 5.21 (dt,J= 52.4, 3.8 Hz, 1H), 4.53 -4.33 (m, 2H ), 4.21 (t,J= 5.8 Hz, 2H), 4.00 (q,J= 4.9 Hz, 1H), 2.28 (t,J= 20.4 H<z>, 2H), 1.93 (<s>, 2H), 1.74 - 1.47 (m, 7H). ESI MS [M-Hf for C17H24CIFN4O8P2, cal. 527.1, found 527.2.

Reference Example 140

Synthesis of acid [({[(2R,3R,4S,5R)-5-{6-chloro-4-[cidopentyl(methyl)amino]-1H-imidazo[4,5-c]pyridin-1-yl} -4-fluoro-3-hydroxyoxolan-2-M]methoxy}(hydroxy)phosphoryl)-methyl]phosphonic

[0513]

[0514] The base compound was synthesized in a similar manner to Example 139. 1H NMR (400 MHz, DMSO-d6) 6 8.24 (d,J= 1.8 Hz, 1H), 6.98 (d,J= 0.7 Hz, 1H), 6.39 (dd,J= 15.4, 4.4 Hz, 1H), 5.80 (p,J= 7.7 Hz, 1H), 5.41 - 5.03 (<m>, 1H), 4.43 (ddd,J= 19.9, 5.4, 3.5 Hz, 1H), 4.31 -4.14 (m, 2H), 4.01 (q,J= 4.9 Hz, 1H), 3.15 (<s>, 4H), 2.29 (t,J= 20.5 H<z>, 2H), 1.94 1.47 (m, 9H). ESI MS [M-H]- for C18H26CIFN4O8P2, cal. 541.1, found 541.2.

Table 1: Specific Examples (Power: IC50 of CD73: means > 1 j M, + means 100 Nm to 1 j M, ++ means < 100 Nm).

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Biological Examples

Materials and methods

[0515] The following general materials and methods were used, where indicated, or may be used in the Examples below: Standard methods in molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, VoIs 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (VoI. 1), cloning in mammalian and yeast cells (VoI.2), Io<s>glucoconjugates and protein expression (VoI. 3), and bioinformatics (VoI.4)).

[0516] The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins and protein glycosylation (see, for example , Coligan, et al. (2000) Current Protocols in Protein Science, VoIs. 1-2, John Wiley and Sons, Inc., NY).

[0517] There are software packages and databases to determine, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites and sequence alignments (see, for example, GCG Wisconsin Package (Accelrys, Inc ., San Diego, CA); and DeCypher™ (TimeLogic Corp., Crystal Bay, NV).

[0518] The literature is full of tests and other experimental techniques that can serve as a basis for the evaluation of the compounds described here.

[0519] Inhibition of Ecto-5'-nucleotidase activity. Io<s>compounds were evaluated for their ecto-5'-nucleotidase (CD73) inhibitory activity. Briefly, CHO-K1 cells stably transfected with human CD73 were generated by LakePharma (Belmont, CA) using molecular cloning of human CD73 (http://www.uniprot.org/uniprot/P21589) and expression vector mammalian transient (P21589.1). After antibiotic selection in CD OptiCHO cell medium (Invitrogen, Catalog n012681-011) containing 5 pg/mL of puromycin and 200 pg/mL of hygromycin B, a suspension of CHO-CD73 cells was collected and frozen in DMSO at 7 .5% in cell medium without antibiotics.

[0520] On the day of the experiment, a vial of CHO-CD73 cells was thawed and suspended in assay medium consisting of 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCOs and 0.1% glucose. To test the ability of Io<s>compounds to inhibit the enzymatic activity of CD73, 2 pL of 500 pM of compounds dissolved in DMSO (50<x>) were added to a 96-well polystyrene plate containing 58 pL of buffer of testing. Next, 20 pL of CHO-CD73 cells in assay buffer was added to the assay plate, followed by 20 pL of 125 pM AMP (adenosine 5'-monophosphate monohydrate) in assay buffer. Final assay conditions consisted of 2500 cells per well in 2% DMSO and 25 pM AMP substrate. After 50 minutes of incubation (37 °C and 5% CO2) and centrifugation at 225*g for 5 minutes, 80 μl of supernatant was transferred to a 96-well Spectra plate (PerkinElmer, cat. no. 6005640) that was previously dispensed. with 20 μl of PiColorLock Gold colorimetric assay reagents (Thermo, cat. no. 3030030). The amount of inorganic phosphate was determined by reading the absorbance at 620 nm on an EnVision Multilabel Plate Reader (PerkinElmer). The enzymatic activity of CD73 was based on the amount of phosphate generated. The percentage of activity was calculated from Io<s>control wells with DMSO and without cells. The IC50 values of the compounds were determined by non-linear regression fitting of four parameters of the percentage of activity in the GraphPad Prism software.

[0521] Pharmacodynamic and pharmacokinetic evaluation. A pharmacodynamic assay may be based on the measurement of CD73-mediated serum adenosine levels. Adenosine levels can be determined by HPLC analysis, and serum compound levels can also optionally be determined in the same HPLC analysis.

[0522] Particular embodiments of this invention are described herein, including the best way known to the inventors to carry out the invention. Upon reading the above description, variations of the disclosed embodiments may become apparent to persons working in the art, and it is expected that skilled craftsmen may employ such variations as appropriate. Accordingly, it is intended that the invention be practiced other than as specifically described herein, and that the invention be defined by the claims appended hereto.

Claims (20)

r e iv in d i c a t i o n s 1. A compound that has the formula: or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein, each R<1> is independently selected from the group consisting of hydrogen, optionally substituted Ci -C6 alkyl, optionally substituted aryl and -C(R<2>R<2>)-0-C (0)-0R<3>, or two R<1>groups are optionally combined to form a 5- to 7-membered ring; each R<2>is independently selected from the group consisting of H and optionally substituted C1-C6 alkyl; each R<3>is independently selected from the group consisting of H, C1-C6 alkyl and optionally substituted aryl; R<5>is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is selected from the group consisting of O, CH2 and S; A is selected from the group formed by: integer between 0 and 3; Z is selected from the group consisting of O, CH2, CHR6 and NR6; each R6 is independently selected from the group consisting of OH, H, CH3, CN, F, optionally substituted C1-C6 alkyl and 0C(0)-C1-C6 alkyl; and optionally two R6 groups at adjacent ring vertices are joined to form a 5- to 6-membered ring having at least one heteroatom as a ring vertex; and Het is selected from the group consisting of: in which the wavy line indicates the point of union with the rest of the compound, and in which: Ra is selected from the group consisting of H, NH2, NHR<7>, NHC(0)R<7>, NR<7>R<7>, R<7>, OH, SR<7>and OR<7> ; Rb is selected from the group consisting of H, halogen, NH2, Nh R7, NR<7>R<7>, R<7>, OH, and OR<7>; Rc is selected from the group consisting of H, halogen, haloalkyl, NH2, NHR<7>, NR<7>R<7>, R<7>, OH, OR<7>, SR<7>, SO2R7, - X<1>-NH2, -X<1>-NHR<7>, -X<1>-NR<7>R<7>, -X<1>-OH, -X<1>-OR<7 >, -X<1>-SR<7>and -X<1>-S0<2>R<7>; Re is selected from the group consisting of H, halogen and optionally substituted C1-C6 alkyl; each X<1>is C1-C4 alkylene; and each R<7>is independently selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C2-C10 alkenyl, optionally substituted C2-C10 alkyne, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkyl, cycloheteroalkyl 4-7 membered optionally substituted, cycloheteroalkylC1-C4alkyl optionally substituted 4-7 membered alkyl, optionally substituted aryl, arylC1-C4alkyl optionally substituted, arylC2-C4alkenyl optionally substituted, arylC2-C4alkynyl optionally substituted, heteroaryl optionally substituted, heteroarylC1-C4alkyl optionally substituted , optionally substituted heteroarylC1-C4alkenyl, optionally substituted heteroarylC2-C4alkynyl and, optionally, two R<7> groups attached to a nitrogen atom join to form a 4- to 7-membered heterocyclic ring, optionally fused to an aryl ring. 2. The compound of claim 1, wherein A is: which is optionally replaced with 1 to 5 R6; optionally, where A is selected from the group consisting of: 3. The compound of claims 1 or 2, wherein R5 is H, X is O, and each R1 is H. 4. The compound of claim 1 or claim 2, wherein R5 is H, X is O, each R1 is H, Re is H, and Ra is selected from the group consisting of NH2, NHR7 and N(R7 )2. 5. The compound of claim 1 or claim 2, wherein R5 is H, X is O, each R1 is H, Re is H, Rc is other than H, and Ra is NHR7. 6. The compound of any of claims 1 to 5, wherein HET is 7. The compound of any of claims 1 to 5, wherein HET is 8. The compound of any of claims 1 to 5, wherein Het-A- is either 9. The compound of any of claims 1 to 5<u>8, wherein R<c>is halo. 10. The compound of any of claims 1 to 5 or 8, wherein Ra is NHR7. 11. The compound of claim 1, selected from: 12. The compound of claim 1 which is either or a pharmaceutically acceptable salt, hydrate or solvate thereof. 13. The compound according to claim 1 which is or a pharmaceutically acceptable salt thereof. 14. A pharmaceutical composition comprising a compound of any of claims 1 to 13 or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable excipient. 15. The compound of any of claims 1 to 13, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in a method of treating a disease, disorder or condition, mediated at least in part by CD73; optionally wherein: said compound is administered in an amount effective to reverse or stop the progression of CD73-mediated immunosuppression. 16. The compound for the use of claim 15, wherein said disease, disorder or condition is: A: cancer, for example, wherein said cancer is a cancer of the prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testicles, head, neck, skin (including melanoma and basal cell carcinoma) lining mesothelial, white blood cells (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell lung carcinoma and non-small cell carcinoma), adrenal gland, thyroid, kidney or bone; or is glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma (including Kaposi's sarcoma), choriocarcinoma, cutaneous basal cell carcinoma, or testicular seminoma; either wherein said cancer is selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, a brain tumor, lymphoma, ovarian cancer and Kaposi's sarcoma; either B: a disease, disorder or condition related to the immune system selected from the group consisting of rheumatoid arthritis, kidney failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, fibromyalgia anemia, Alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infections, Crohn's disease, ulcerative colitis, allergic contact dermatitis and other eczemas, systemic sclerosis and multiple sclerosis. 17. A combination comprising a compound of any of claims 1 to 13, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one additional therapeutic agent; optionally, wherein the at least one additional therapeutic agent is: (i) a chemotherapeutic agent, an immunomodulatory and/or anti-inflammatory agent, an antihypercholesterolemia agent or an anti-infective agent; either (ii) an immune checkpoint inhibitor. 18. A kit comprising a compound of any of claims 1 to 13, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one additional therapeutic agent; optionally in which (i) the at least one additional therapeutic agent is a chemotherapeutic agent, an immunomodulatory and/or anti-inflammatory agent, an antihypercholesterolemia agent or an anti-infective agent; either (ii) the at least one additional therapeutic agent is an immune checkpoint inhibitor. 19. The compound of any of claims 1 to 13, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in a method of treating cancer in a subject, said method comprising administering to said subject an effective amount of the compound and an immune checkpoint inhibitor; optionally: (i) wherein said administration is prior to, simultaneous with, or subsequent to radiotherapy; I (í) wherein said compound and said immune checkpoint inhibitor are administered in combination; or (<í>¡) wherein said compound and said immune checkpoint inhibitor are administered sequentially; or (v) wherein said compound is administered after said immune checkpoint inhibitor; either (v) wherein said compound is administered before said immune checkpoint inhibitor. 20. The combination, kit or compound for use of any of claims 17, 18 or 19, wherein said immune checkpoint inhibitor: a) inhibits PD-1, PD-L1 or CTLA-4; either b) is selected from the group consisting of ipilimumab, nivolumab and lambrolizumab.